Transformed cell with enhanced sensitivity to antifungal compound and use thereof

ABSTRACT

The present invention provides a transformed cell in which a polynucleotide having a nucleotide sequence encoding an amino acid sequence of an osmosensing histidine kinase having no transmembrane region is introduced in a functional form into a cell deficient in at least one hybrid-sensor kinase, a method of assaying the antifungal activity of a test substance using the transformed cell, and a method of searching an antifungal compound using the method, and the like.

CROSS-REFERENCED INFORMATION

This application is a Divisional of U.S. application Ser. No.12/272,705, filed Nov. 17, 2008 now U.S. Pat. No. 7,759,084; which is aDivisional of U.S. application Ser. No. 10/697,036 filed Oct. 31, 2003(now U.S. Pat. No. 7,452,688), which claims priority of JapaneseApplication No. 2002-317736 filed Oct. 31, 2002. The entire disclosuresare hereby incorporated by reference of its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a transformed cell with enhancedsensitivity to an antifungal compound and use thereof.

2. Description of the Related Art

It is known that, when a fungicide containing a dicarboxyimideantifungal compound, an “aromatic hydrocarbon antifungal compound” or aphenylpyrrole antifungal compound as an active ingredient is acted on acertain plant-pathogenic filamentous fungus, glycerol synthesis in acell is stimulated in the fungus like as when undergoes high osmoticstress, and the fungus can not control an intracellular osmolarity,leading to death. From such the activity to the plant-pathogenicfilamentous fungus, a protein in a signal transduction system which isinvolved in osmolarity response was predicted as a target protein of anantifungal compound contained in these fungicides as an activeingredient.

In Neurospora crassa exhibiting sensitivity to the aforementionedantifungal compound, an osmosensitive mutant os-1 was reported. Thismutant os-1 exhibited resistance to the aforementioned antifungalcompound and, by analysis of the mutant, an os-1 gene which is anosmosensing histidine kinase gene was isolated as a causative gene. Aprotein having an amino acid sequence encoded by a nucleotide sequenceof this os-1 gene was a protein which has a structure of histidinekinase of a two-component regulatory system and, at the same time, has acharacteristic region (hereinafter, referred to repeat sequence regionin some cases) in which amino acid sequences composed of about 90 aminoacids and having homology to each other are present repetitively about 6times (see, for example, U.S. Pat. No. 5,939,306; Genebank accessionU50263, U53189, AAB03698, AAB01979; Alex, A. L. et al., Proc. Natl. Acd.Sci. USA 93:3416-3421; Schumacher, M. M. et al., Current Microbiology34:340-347; Oshima, M. et al., Phytopathology 92 (1):75-80; Fijimura, M.et al., J. Pesticide Sci. 25:31-36). A gene having homology to the os-1gene was also isolated from plant-pathogenic filamentous fungus such asBotryotinia fuckeliana, Magnaporthe grisea, Fusarium solani and thelike, and its nucleotide sequence and an amino acid sequence encoded bythe gene are published. It is known that genes having homology with theos-1 gene are specifically present in filamentous fungus amongeukaryotic organisms (see, for example, GeneBank accession AF396827,AF435964, AAL37947, AAL30826; Fujimura, M. et al., Pesticide Biochem.Physiol. 67:125-133; GeneBank accession AB041647, BAB40497).

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method of detectingthe antifungal activity and a method of selecting an antifungal compoundusing the os-1 gene and a gene having homology with the gene.

Under such the circumstances, the present inventor intensively studiedand, as a result, found a transformed cell with enhanced sensitivity toan antifungal compound, and found a method of detecting the antifungalactivity using this transformed cell and a method of selecting anantifungal compound using this transformed cell, which resulted incompletion of the present invention.

Thus, the present invention provides:

A transformed cell in which a polynucleotide encoding an osmosensinghistidine kinase having no transmembrane region is introduced in afunctional form into a cell that is deficient in at least onehybrid-sensor kinase, wherein the osmosensing histidine kinase having notransmembrane region has been obtained from Fusarium oxysporum,Mycospharella tritici or Thanatephorus cucumeris.

The transformed cell according to claim 1, wherein the polynucleotidecomplements the hybrid-sensor kinase deficiency.

The transformed cell according to claim 1, wherein the cell is amicroorganism cell.

The transformed cell according to claim 1, wherein the cell is a buddingyeast cell.

The transformed cell according to claim 1, wherein the osmosensinghistidine kinase having no transmembrane region has an amino acidsequence selected from the group consisting of:

(a) an amino acid sequence of an osmosensing histidine kinase having notransmembrane region, which has amino acid sequence homology of 95% ormore to the amino acid sequence of any of SEQ ID NOs: 41, 55 and 68;

(b) an amino acid sequence of an osmosensing histidine kinase having notransmembrane region, which is encoded by a DNA amplified by apolymerase chain reaction using a cDNA obtained from Fusarium oxysporumas a template and using as primers an oligonucleotide comprising thenucleotide sequence of SEQ ID NO: 52 and an oligonucleotide comprisingthe nucleotide sequence of SEQ ID NO: 53;

(c) an amino acid sequence of an osmosensing histidine kinase having notransmembrane region, which is encoded by a DNA amplified by apolymerase chain reaction using a cDNA obtained from Mycospharellatritici as a template and using as primers an oligonucleotide comprisingthe nucleotide sequence of SEQ ID NO: 64 and an oligonucleotidecomprising the nucleotide sequence of SEQ ID NO: 65;

(d) an amino acid sequence of an osmosensing histidine kinase having notransmembrane region, which is encoded by a DNA amplified by apolymerase chain reaction using a cDNA obtained from Thanatephoruscucumeris as a template and using as primers an oligonucleotidecomprising the nucleotide sequence of SEQ ID NO: 85 and anoligonucleotide comprising the nucleotide sequence of SEQ ID NO: 86;

(e) the amino acid sequence of SEQ ID NO: 41;

(f) the amino acid sequence of SEQ ID NO: 55; and

(g) the amino acid sequence of SEQ ID NO: 68.

The transformed cell according to claim 1, wherein the osmosensinghistidine kinase having no transmembrane region has the amino acidsequence of any of SEQ ID NOs: 41, 55 and 68.

The transformed cell according to claim 1, wherein the polynucleotidehas the nucleotide sequence of any of SEQ ID NOs: 42, 56 and 69.

A method of assaying the antifungal activity of a substance, whichcomprises:

(1) culturing the transformed cell as defined in claim 1 in the presenceof a test substance;

(2) measuring an amount of intracellular signal transduction from theosmosensing histidine kinase having no transmembrane region or an indexvalue having the correlation therewith; and

(3) assessing the antifungal activity of the test substance based on adifference between an amount of intracellular signal transduction or anindex value having the correlation therewith measured in (2) and acontrol.

The method of assaying according to claim 8, wherein the amount ofintracellular signal transduction from the osmosensing histidine kinasehaving no transmembrane region or the index value having the correlationtherewith is an amount of growth of the transformed cell.

A method of searching for a potent antifungal compound, which comprisesselecting an antifungal compound based on the antifungal activityassessed in the assaying method as defined in claim 8.

A polynucleotide encoding an amino acid sequence selected from the groupconsisting of:

(a) an amino acid sequence of an osmosensing histidine kinase having notransmembrane region, which has amino acid sequence homology of 95% ormore to the amino acid sequence of any of SEQ ID NOs: 41, 55 and 68;

(b) an amino acid sequence of an osmosensing histidine kinase having notransmembrane region, which is encoded by a DNA amplified by apolymerase chain reaction using a cDNA obtained from Fusarium oxysporumas a template and using as primers an oligonucleotide comprising thenucleotide sequence of SEQ ID NO: 52 and an oligonucleotide comprisingthe nucleotide sequence of SEQ ID NO: 53;

(c) an amino acid sequence of an osmosensing histidine kinase having notransmembrane region, which is encoded by a DNA amplified by apolymerase chain reaction using a cDNA obtained from Mycospharellatritici as a template and using as primers an oligonucleotide comprisingthe nucleotide sequence of SEQ ID NO: 64 and an oligonucleotidecomprising the nucleotide sequence of SEQ ID NO: 65;

(d) an amino acid sequence of an osmosensing histidine kinase having notransmembrane region, which is encoded by a DNA amplified by apolymerase chain reaction using a cDNA obtained from Thanatephoruscucumeris as a template and using as primers an oligonucleotidecomprising the nucleotide sequence of SEQ ID NO: 85 and anoligonucleotide comprising the nucleotide sequence of SEQ ID NO: 86;

(e) the amino acid sequence of SEQ ID NO: 41;

(f) the amino acid sequence of SEQ ID NO: 55; and

(g) the amino acid sequence of SEQ ID NO: 68.

The polynucleotide according to claim 11, which encodes the amino acidsequence of any of SEQ ID NOs: 41, 55 and 68.

The polynucleotide according to claim 11, which has the nucleotidesequence of any of SEQ ID NOs: 42, 56 and 69.

A method of obtaining from a plant-pathogenic filamentous fungus apolynucleotide encoding an osmosensing histidine kinase having notransmembrane region, which comprises:

amplifying a desired polynucleotide by polymerase chain reaction usingas a primer an oligonucleotide comprising the nucleotide sequence of anyof SEQ ID NOs: 52, 53, 64, 65, 85 and 86, and recovering the amplifieddesired polynucleotide.

The method, wherein the plant-pathogenic filamentous fungus may beFusarium oxysporum, and wherein the desired polynucleotide is amplifiedusing as primers an oligonucleotide comprising the nucleotide sequenceof SEQ ID NO: 52 and an oligonucleotide comprising the nucleotidesequence of SEQ ID NO: 53.

The method, wherein the plant-pathogenic filamentous fungus may beMycospharella tritici, and wherein the desired polynucleotide isamplified using as primers an oligonucleotide comprising the nucleotidesequence of SEQ ID NO: 64 and an oligonucleotide comprising thenucleotide sequence of SEQ ID NO: 65.

The method, wherein the plant-pathogenic filamentous fungus may beThanatephorus cucumeris, and wherein the desired polynucleotide isamplified using as primers an oligonucleotide comprising the nucleotidesequence of SEQ ID NO: 85 and an oligonucleotide comprising thenucleotide sequence of SEQ ID NO: 86.

An osmosensing histidine kinase having no transmembrane region, which isencoded by the polynucleotide as defined in claim 10.

The osmosensing histidine kinase having no transmembrane region which isencoded by the polynucleotide as defined in claim 10, which has theamino acid sequence of any of SEQ ID NOs: 41, 55 and 68

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The present invention will be explained in detail below.

The “transformed cell in which a polynucleotide having a nucleotidesequence encoding an amino acid sequence of an osmosensing histidinekinase having no transmembrane region is introduced in a functional forminto a cell deficient in at least one hybrid-sensor kinase” is obtainedby introducing a polynucleotide having a nucleotide sequence encoding anamino acid sequence of an “osmosensing histidine kinase having notransmembrane region” in a functional form into a “cell deficient in atleast one hybrid-sensor kinase” which is a host cell. Herein,“introduction of a polynucleotide in a functional form” means that thepolynucleotide is introduced so as to complement the deficiency inhybrid-sensor kinase, in other words, that the polynucleotide isintroduced in such a form that a phenotype of the cell caused by thedeficiency in hybrid-sensor kinase revert to a phenotype without thedeficiency in hybrid-sensor kinase. Specifically, for example, in thecase of budding yeast (e.g. Saccharomyces cerevisiae), when SLN1 whichis hybrid-sensor kinase is deleted, the SLN1-deficient yeast cell showsa phenotype that the cell can not grow under the normal growingcondition. By introducing a polynucleotide having a nucleotide sequenceencoding an amino acid sequence of SLN1 isolated from budding yeast intothe SLN1-deficient cell so that SLN1 is expressed (e.g. operably linkedto downstream of a promoter), the cell becomes possible to grow underthe normal growing condition. The “cell deficient in at least onehybrid-sensor kinase” may be obtained, for example, by deleting at leastone intrinsic hybrid-sensor kinase. First, hybrid-sensor kinase will beexplained below.

(Two-Component Regulatory System and Hybrid-Sensor Kinase)

Two-component regulatory system is a signal transduction system which iswidely used in prokaryotic organisms and, since this system is basicallycomposed of two proteins called a sensor and a regulator, it is calledtwo-component regulatory system. In a typical two-component regulatorysystem, a sensor is composed of an input region and a histidine kinaseregion, and a regulator is composed of a receiver region and an outputregion. When the input region senses an environmental stimulus, ahistidine residue in an amino acid sequence in the histidine kinaseregion which is well conserved among organisms is phosphorylated ordephosphorylated. Herein, phosphorylation of the histidine residue isautophosphorylation utilizing ATP as a substrate. This phosphate groupis transferred to an aspartic acid residue in an amino acid sequence inthe receiver region in the regulator which is well conserved amongorganisms, and phosphorylation and dephosphorylation of the asparticacid residue regulates the activity of the output region in theregulator. In the case of prokaryotic organisms, the output region is atranscription regulating factor in many cases although there areexceptions, and the regulator directly controls gene expression throughthe aforementioned phosphoryl transfer in response to stimuli sensed bythe sensor.

A sensor takes a more complicated structure in some cases unlike theaforementioned typical structure. For example, in addition to astructure composed of an input region and a histidine kinase region,following this, the sensor contains a receiver region, which is observedin a regulator, on its C-terminal side in some cases. In this case, thephosphorylay system of a phosphate group becomes more complicated, andit is known that a phosphate is transferred from the sensor to aregulator called a response regulator via an intervening protein havinga transmitter region called a phosphotransmitter. That is, when theinput region of the sensor senses stimuli, phospate is transferred tomediate signal transduction from a histidine residue of the histidinekinase region in the same molecule to an aspartic acid residue of thereceiver region in the same molecule, then, to a histidine residue ofthe phosphotransmitter, finally, to an aspartic acid residue of thereceiver region in a response regulator. Like this, two-componentregulatory system is associated with three proteins in some cases. Suchthe sensor involved in signal transduction system through phosphoryltransfer composed of three proteins and having the aforementionedstructural characteristic is referred to as “hybrid-sensor kinase”.Hybrid-sensor kinase is found not only in a prokaryotic organism butalso in an eukaryotic microorganism such as yeast, a plant and the like,and is involved in response to a variety of stimuli or stresses.

Herein, an input region of a hybrid-sensor kinase is a region present atthe N-terminal of the kinase, and have a transmembrane region in manycases. The transmembrane region can be revealed by a structureprediction analysis using a structure prediction software, for example,TMpred program [K. Hofmann & W. Stoffel, Biol. Chem. Hoppe-Seyler, 374,166 (1993)] which is available, for example, fromhttp://www.ch.embnet.org/software/TMPRED_form.html. A histidine kinaseregion of a hybrid-sensor kinase is, for example, a region following theC-terminal of the input region, and is a region characterized in that ithas five conserved motifs common to general histidine kinases asdescribed in Parkinson, J. S. & Kofoid, E. C. (1989) Annual Review ofGenetics 23:311-336, Stock, J. B. et. al. (1989) Microbiological Reviews53(4):450-490. For example, in the hybrid-sensor kinase SLN1 of buddingyeast, a histidine kinase region is the region from amino acid residues556 to 908. A receiver region of a hybrid-sensor kinase is, for example,a region following the C-terminal of the histidine kinase region, and isa region characterized in that it has three conserved motifs common togeneral histidine kinases as described in Parkinson, J. S. & Kofoid, E.C. Annual Review of Genetics 23:311-336 (1989), Stock, J. B. et. al.(1989) Microbiological Reviews 53(4): 450-490. For example, in thehybrid-sensor kinase SLN1 of budding yeast, a receiver region is theregion from amino acid residues 1088 to 1197.

As a signal transduction system after a response regulator, in additionto a simple system in which an output region of a regulator is atranscription regulating factor as described above, as a morecomplicated system, there is known a system in which a signal istransmitted to a transcription regulating factor participating incontrol of gene expression, via MAP kinase cascade which is associatedwith various controls in a cell.

Specific examples of a hybrid-sensor kinase and a signal transductionsystem which involves the hybrid-sensor kinase will be explained below.

(Hybrid-Sensor Kinase of Budding Yeast)

In budding yeast (Saccharomyces cerrevisiae), the hybrid-sensor kinaseSLN1 is utilized for signal transduction relating to osmolarityresponse. The SLN1 is a sole histidine kinase found in budding yeast.SLN1 is an osmosensing histidine kinase having a transmembrane region inits input region, and mediates a phosphoryl transfer signal to theresponse regulator SSK1 via the phosphotransmitter YPD1. Downstream ofthe signal transduction, MAP kinase cascade composed of three kinasesSSK2 (MAPKKK), PBS2 (MAPKK) and HOG1 (MAPK) lies to regulate expressionof genes involved in osmolarity adaptation such as glycerol biosynthesisand the like. The output region of the response regulator SSK1 has anactivity of phosphorylating SSK2. The SSK1 is negatively controled byphosphorylation of an aspartic acid residue in its receiver region, thephosphorylating activity of whose output region is inhibited.Specifically, at a normal osmolarity, a histidine residue in thehistidine kinase region of SLN1 is autophosphorylated, and the phosphateis subsequently transferred to an aspartic acid residue of the receiverregion in the same molecule, then, to a histidine residue of YPD1,finally, to an aspartic acid residue in the receiver region of SSK1. Byphosphorylation of an aspartic acid residue in the receiver region ofSSK1, the phosphorylating activity of the output region of SSK1 issuppressed, and the phosphate is not transferred to a MAP kinase cascadecomposed of SSK2, PBS2 and HOG1, and then expression of genes involvedin osmolarity adaptation such as glycerol biosynthesis and the like arenot induced. On the other hand, under a condition of high osmolarity,since autophosphorylation of a histidine residue of the histidine kinaseregion is inhibited in SLN1, the MAP kinase cascade composed of SSK2,PBS2 and HOG1 is activated, and then expression of genes involved inosmolarity adaptation such as glycerol biosynthesis and the like isinduced (Maeda, T. et. al. (1994) Nature 369:242-245).

(Hybrid-Sensor Kinase of Fission Yeast)

In fission yeast (Scchizosaccharomyces pombe), three kinds ofhybrid-sensor kinases PHK1 (MAK2), PHK2 (MAK3) and PHK3 (MAK1)participate in regulation of cell cycle progression [G(2) to M phasetransition] and oxidative stress response. In a fission yeast, there isno histidine kinase other than PHK1, PHK2 and PHK3. PHK1 and PHK2 arehistidine kinases responsive to oxidative stress such as hydrogenperoxide and the like (Buck, V. et. al., Mol. Biol. Cell 12:407-419).Three kinds of hybrid-sensor kinases PHK1, PHK2 and PHK3 metiate aphosphoryl transfer signal to the response regulator MCS4 via thephosphotransmitter SPY1 (MPR1). Downstream of this signal transduction,a MAP kinase cascade composed of three kinases WAK1 (MAPKKK), WIS1(MAPKK) and STY1 (MAPK) lies to regulate expression of genes involved inregulation of cell cycle progression and oxidative stress response. Theoutput region of the response regulator MCS4 has an activity ofphosphorylating WAK1. The MCS4 is negatively controled byphosphorylation of an aspartic acid residue in its receiver region, thephosphorylating activity of whose output region is inhibited.Specifically, under a normal condition, each of histidine residues inthe histidine kinase regions of PHK1 to PHK3 is autophosphorylated, andthe phosphates are transferred to each of aspartic acid residues ofreceiver regions in the same molecule, then, to a histidine residue ofSPY, finally, to an aspartic acid residue in the receiver region ofMCS4. By phosphorylation of an aspartic acid residue in the receiverregion of MCS4, the phosphorylating activity of the output region ofMCS4 is suppressed, and the phosphate is not transferred to a MAP kinasecascade composed of WAK1, WIS1 and STY1, and then expression of genesinvolved in regulation of cell cycle progression and stress response arenot induced. On the other hand, under a stress condition,autophosphorylation of each of histidine residues of the histidinekinase regions in PHK1 to PHK3 is inhibited, a MAP kinase cascadecomposed of WAK1, WIS1 and STY1 is activated, and expression of genesinvolved in control of cell cycle progression and oxidative stressresponse are induced. As a result, it is observed such a phenotype thatG(2) to M phase transition in cell cycle progression of the fissionyeast is promoted, and that a dividing cell length becomes remarkablyshorter than usual (Aoyama, K. et. al. (2001) Boisci. Biotechnol.Biochem. 65:2347-2352).

(Hybrid-Sensor Kinase of Bacterium)

In a prokaryotic organism Escherichia coli, the hybrid-sensor kinaseRcsC participates in control of expression of the cps operon involved incapsular polysaccharide synthesis. RcsC is a histidine kinase having atransmembrane region, and it is known that it mediates a phosphoryltransfer signal to the response regulator RcsB via thephosphotransmitter YojN. The output region of RcsB has an activity ofinducing transcription of the cps operon. Specifically, under a normalcondition, a histidine residue in the histidine kinase region of RcsC isautophosphorylated, and the phosphate is transferred to an aspartic acidresidue of the receiver region in the same molecule, then, to ahistidine residue of YojN, finally, to an aspartic acid residue in thereceiver region of RcsB. By phosphorylation of an aspartic acid residuein the receiver region of RcsB, the cps operon transcription inducingactivity of the output region of RcsB is suppressed, and expression ofgenes involved in capsular polysaccharide synthesis are not induced. Onthe other hand, under a condition of high osmolarity, in RcsC,autophosphorylation of a histidine residue in the histidine kinaseregion is inhibited, the cps operon transcription inducing activity ofthe output region of RcsB is activated, and expression of genes involvedin capsular polysaccharide synthesis are induced (Clarke, D. J. et. al.(2002) J. Bactriol. 184:1204-1208).

A bioluminescent marine microorganism Vibrio harveyi emits fluorescentlight generated in luciferase reaction depending on its own celldensity. Hybrid-sensor kinases LuxN and LuxQ parcipite in control ofexpression of a gene involved in this cell density-responsivebioluminescence. LuxN and LuxQ are histidine kinases each having atransmembrane region. To sense its own cell density, V. harveyi producesand secrets two kinds of substances (AI-1, AI-2) called autoinducer.AI-1 is sensed by LuxN and AI-2 is sensed by LuxQ to convey cell-densityinformation. LuxN and LuxQ mediate phosphoryl transfer signals to theresponse regulator LuxO via the phosphotransmitter LuxU. The outputregion of LuxO has an activity of inducing transcription of theluciferase operon. To specifically explain by referring to LuxN, when acell density is low, since AI-1 in the environment is at low level andis not sensed by the input region of LuxN, a histidine residue in thehistidine kinase region of LuxN is autophosophorylated. The phosphate istransferred to an aspartic acid residue of the receiver region in thesame molecule, then, to a histidine residue of LuxU, finally, to anaspartic acid residue in the receiver region of LuxO. By phosphorylationof an aspartic acid residue in the receiver region of LuxO, theluciferase operon transcription inducing activity of the output regionof LuxO is suppressed, and expression of genea involved inbioluminescence are not induced. On the other hand, under a high celldensity condition, since AI-1 in environment is at high level and issensed by the input region of LuxN, autophosphorylation of a histidineresidue of the histidine kinase region is inhibited in LuxN, theluciferase operon transcription inducing activity of the output regionof LuxO is activated, and bioluminescence is induced (Freeman, J. A. et.al. (2000) Mol. Microbiol. 35:139-149).

(Hybrid-Sensor Kinase of Plant)

In a higher plant Arabadopsis thaliana, receptor proteins CRE1, AHK2 andAHK3 for a plant hormone cytokinin are hybrid-sensor kinases. Receptorproteins CRE1, AHK2 and AHK3 are all cytokinin-sensitive histidinekinase having a transmembrane region (Inoue, T. et. al. (2001) Nature409:1060-1063). CRE1 mediates a phosphoryl transfer signal to responseregulators ARR1, ARR2 and ARR10 via phosphotransmitters AHP1 and AHP2.It is considered that output regions of ARR1, ARR2 and ARR10 have anactivity of inducing transcription of cytokinin-inducing genes ARR4 toARR7. Specifically, in the presence of cytokinin, a hisitidine residuein the histidine kinase region of CRE1 is autophosphorylated, and thephosphate is transferred to an aspartic acid residue of the receiverregion in the same molecule, then, to histidine residues of AHP1 andAHP2, finally, to aspartic acid residues in receiver regions of ARR1,ARR2 and ARR10. By phosphorylation of aspartic acid residues in receiverregions of ARR1, ARR2 and ARR10, a gene transcription inducing activityof output regions of ARR1, ARR2 and ARR10 are promoted, and expressionof cytokinin-responsive genes ARR4 to 7 is induced (Hwang, I. & Sheen J.(2001) Nature 413:383-389).

(Cell Deficient in at Least One Hybrid-Sensor Kinase)

“The cell deficient in at least one hybrid-sensor kinase” means a cellin which function of at least one intrinsic hybrid-sensor kinase islost. Examples of the cell include a cell in which production of atleast one intrinsic hybrid-sensor kinase is deleted, suppressed orinhibited, a cell in which activity of at least one intrinsichybrid-sensor kinase is deleted, suppressed or inhibited, and the like.More specific examples include budding yeast deficient in SLN1, fissionyeast deficient in all of three of PHK1, PHK2 and PHK3, Escherichia colideficient in RcsC, V. harveyi deficient in LuxN, Arabidopsis thalianadeficient in CRE1, and the like.

In order to prepare the “cell deficient in at least one hybrid-sensorkinase”, for example, deletion, addition, substitution or the like ofone or more nucleotides are introduced into the whole or a part of apromoter region or a coding region of a gene encoding hybrid-sensorkinase to be deleted. Specifically, for example, the SLN1-deficientbudding yeast strain TM182 can be prepared by the method described inMaeda, T. et. al. (1994) Nature 369:242-245, the PHK1, PHK2 andPHK3-deficient fission yeast strain KI011 can be prepared by the methoddescribed in Aoyama, K. et. al. (2001) Boisci. Biotechnol. Biochem.65:2347-2352. In addition, the RcsC-deficient Escherichia coli strainSRC122 can be prepared by the method described in Suzuki, T., et. al.(2001) Plant Cell Physiol. 42:107-113, and the LuxN-deficient V. harveyistrain BNL63 can be prepared by the method described in Freeman, J. A.et. al. (2000) Mol. Micobiol. 35:139-149. For preparing a CRE1-deficientArabidopsis thaliana, for example, a clone defective in cytokineresponse is selected from clones obtained by mutagenesis of Arabidopsisthaliana according to the method described in Inoue, T. et. al. (2001)Nature 409:1060-1063. Genomic CRE1 gene fragment is amplified by PCRusing a primer designed based on the nucleotide sequence of the genomicCRE1 gene listed in Genebank accession AB049934 and using a genomic DNAof the selected clone as a template, and its nucleotide sequence isconfirmed, whereby, a CRE1-deficient clone which can not express CRE1can be selected.

Alternatively, a cell deficient in unknown hybrid-sensor kinase besidesthe aforementioned kinases may be also prepared, for example, byisolating a hybrid-sensor kinase gene from a desired cell, and deletingthe gene harbored by the cell by homologous recombination using thegene. For isolating a hybrid-sensor kinase gene of a desired cell, thestructural characteristic of hybrid-sensor kinases can be utilized. Forexample, amino acid sequences around the histidine residue to beautophosphorylated are conserved among hisitidine kinase regions andamino acid sequences around the aspartic acid residue to which aphosphate to be transferred from the histidine residue are conservedamong receiver regions. Then, a hybrid-sensor kinase gene of a desiredcell can be isolated by a polymerase chain reaction (hereinafter,referred to as PCR) using an oligonucleotide designed based on anucleotide sequence encoding the aforementioned conserved amino acidsequences as a primer, or a hybridization method using anoligonucleotide having a nucleotide sequence encoding the aforementionedconserved amino acid sequences as a prove. By examining whether or notthe aforementioned structural characteristic is possessed based on anamino acid sequence deduced from a nucleotide sequence of the isolatedgene, it can be confirmed that the isolated gene is a gene having anucleotide sequence encoding an amino acid sequence of a hybrid-sensorkinase. A specific example is a PCR method described in Srilantha, T.et. al. (1998) Microbiology 144:2715-2729. For PCR and hybridization,for example, the experimental conditions using upon isolation of the“polynucleotide having a nucleotide sequence encoding an amino acidsequence of osmosensing hisitidine kinase having no transmembraneregion” described later may be used.

Alternatively, a hybrid-sensor kinase gene may be also isolated using,as an index, the functional complementation in budding yeast in whichexpression of SLN1 is conditionally suppressed, for example, accordingto the method described in Nagahashi, S. et. al. (1998) Microbiology144:425-432.

(Osmosensing Histidine Kinase Having No Transmembrane Region)

Then, the “osmosensing histidine kinase having no transmembrane region”to be introduced into the aforementioned “cell deficient in at least onehybrid-sensor kinase” in a functional form will be explained.

In filamentous fungus, a histidine kinase having a structure similar tothat of the aforementioned hybrid-sensor kinase is isolated. Thehistidine kinase has a histidine kinase region and a receiver regionwhich are observed in hybrid-sensor kinases, and has no transmembraneregion, which is observed in many hybrid-sensor kinases, in its inputregion, and further has a characteristic structure in which amino acidsequences composed of about 90 amino acids having the amino acidsequence homology to each other are present repeatedly about six times,in place of the transmembrane region. Although a signal transductionpathway from this histidine kinase has not been completely clarified, itis known that the signal transduction participates in osmolarityresponse.

In the present invention, “homology” refers to identity of sequencesbetween two genes or two proteins. The “homology” is determined bycomparing two sequences aligned in the optimal state, over a region of asequence of a subject to be compared. Herein, in optimal alignment ofnucleotide sequences or amino acid sequences to be compared, addition ordeletion (e.g. gap etc.) may be allowable. Such the “homology” can becalculated by homology analysis with making alignment using a program ofFASTA [Pearson & Lipman, Proc. Natl. Acad. Sci. USA, 4, 2444-2448(1998)], BLAST [Altschul et. al. Journal of Molecular Biology, 215,403-410 (1990)], CLUSTAL W [Thompson, Higgins & Gibson, Nucleic AcidResearch, 22, 4673-4680(1994a)] and the like. The above programs areavailable to the public, for example, in homepage(http://www.ddbj.nig.ac.jp) of DNA Data Bank of Japan [international DNAData Bank managed in Center for Information Biology and DNA Data Bank ofJapan (CIB/DDBJ)). Alternatively, the “homology” may be also obtained byusing commercially available sequence analysis software. Specifically,the homology can be calculated, for example, by performing homologyanalysis with making alignment by the Lipman-Pearson method [Lipman, D.J. and Pearson, W. R., Science, 227, 1435-1441, (1985)] usingGENETYX-WIN Ver. 5 (manufactured by Software Development Co., Ltd.).

Herein, as the “structure in which amino acid sequences composed ofabout 90 amino acids having the amino acid sequence homology to eachother are repeatedly present about six times”, for example, there is arepeat sequence region described in Alex, L. A. et. al. (1996) Proc.Natl. Acad. Sci. USA 93:3416-3421, Ochiai, N. et. al. (2001) Pest Manag.Sci. 57:437-442, Oshima, M. et. al. (2002) Phytopathology 92:75-80 andthe like, and such the structure is present at the N-terminal region ofthe hisitide kinase. The “amino acid sequences composed of about 90amino acids are repeatedly present about six times” include an aminoacid sequence motif composed of about 90 amino acids is repeated fivetimes followed by a sixth truncated repeat sequence (5.7 times repeat),an amino acid sequence motif composed of about 90 amino acids isrepeated six times followed by a seventh truncated repeat sequence (6.7times repeat), and the like. Specifically, in amino acid sequence of ahistidine kinase of the present invention, examples of the “a region inwhich amino acid sequences composed of about 90 amino acids having theamino acid sequence homology to each other are present repeatedly aboutsix times” include a region from amino acid residues 190 to 707 in anamino acid sequence represented by SEQ ID NO: 1 (5.7 times repeat), aregion from amino acid residues 189 to 706 in an amino acid sequencerepresented by SEQ ID NO: 16 (5.7 times repeat), a region from aminoacid residues 176 to 693 in an amino acid sequence represented by SEQ IDNO: 41 (5.7 times repeat), a region from amino acid residues 192 to 709in an amino acid sequence represented by SEQ ID NO: 55 (5.7 timesrepeat), and a region from amino acid residues 299 to 911 in an aminoacid sequence represented by SEQ ID NO: 68 (6.7 times repeat), and thelike.

The “osmosensing histidine kinase having no transmembrane region” is theaforementioned histidine kinase characteristic in filamentous fungus,and refers to a osmosensing protein having a repeat sequence region ofamino acid sequences composed of about 90 amino acids having the aminoacid sequence homology to each other, a histidine kinase region and areceiver region, and having no transmembrane region.

In order to confirm that a protein has the function of osmosensinghistidine kinase, enhancement of the sensitivity of a cell to osmolaritystress may be confirmed when the protein (histidine kinase) is deletedfrom the cell. Alternatively, it may be also confirmed that a protein(histidine kinase) is osmosensing histidine kinase, by confirming thatexpression of the protein in an osmosensing hybrid-sensor kinaseSLN1-deficient budding yeast cell results in a functionalcomplementation of the SLN1 and the budding yeast cell capable ofgrowing.

Among filamentous fungi, mainly, in Neurospora crassa which is a modelorganism of filamentous fungus, a plant pathogenic filamentous funguswhich is a pathogenic microorganism, a host of which is a plant, or thelike, the presence of the “osmosensing histidine kinase having notransmembrane region” is reported.

Examples of the “osmosensing histidine kinase having no transmembraneregion” of the present invention include an osmosensing histidine kinasehaving no transmembrane region, which has an amino acid sequenceselected from the group consisting of:

(a) an amino acid sequence of an osmosensing histidine kinase having notransmembrane region, which has an amino acid sequence homology of 95%or more to the amino acid sequence represented by any of SEQ ID NOs: 41,55 and 68;

(b) an amino acid sequence of an osmosensing histidine kinase having notransmembrane region, which is encoded by a DNA amplified by apolymerase chain reaction using a Fusarium oxysporum-derived cDNA as atemplate and using an oligonucleotide having the nucleotide sequencerepresented by SEQ ID NO: 52 and an oligonucleotide having thenucleotide sequence represented by SEQ ID NO: 53 as primers;

(c) an amino acid sequence of an osmosensing histidine kinase having notransmembrane region, which is encoded by a DNA amplified by apolymerase chain reaction using a Mycospharella tritici-derived cDNA asa template and using an oligonucleotide having the nucleotide sequencerepresented by SEQ ID NO: 64 and an oligonucleotide having thenucleotide sequence represented by SEQ ID NO: 65 as primers;

(d) an amino acid sequence of an osmosensing histidine kinase having notransmembrane region, which is encoded by a DNA amplified by apolymerase chain reaction using a Thanaphthorus cucumeris-derived cDNAas a template and using an oligonucleotide having the nucleotidesequence represented by SEQ ID NO: 85 and an oligonucleotide having thenucleotide sequence represented by SEQ ID NO: 86 as primers;

(e) an amino acid sequence of an osmosensing histidine kinase having notransmembrane region, which is derived from Phytophthora infestans andhas the amino acid sequence represented by SEQ ID NO: 90;

(f) the amino acid sequence represented by SEQ ID NO: 41;

(g) the amino acid sequence represented by SEQ ID NO: 55; and

(h) the amino acid sequence represented by SEQ ID NO: 68.

A preferred amino acid sequence homology in the above (a) may forexample be about 95%, or higher such as about 98%. The difference fromthe amino acid sequence represented by any of SEQ ID: 41, 55 and 68observed in the amino acid sequence of the above (a) may for example bea variation such as the deletion, substitution and addition of aminoacids. Such a variation includes a variation which can artificially beintroduced by means of a site-directed mutagenesis method or a mutagenictreatment as well as a polymorphic variation which occurs naturally suchas a difference in an amino acid sequence resulting from the differenceby the species or strains from which the protein is derived. As thesite-directed mutagenesis method, for example, there is mentioned themethod which utilizes amber mutations (capped duplex method, NucleicAcids Res., 12, 9441-9456 (1984)), the method by PCR utilizing primersfor introducing a mutation and the like.

At least one, specifically one to several (herein “several” means about2 to about 10), or more amino acid residues may be varied in the abovevariations. The amino acid residues may be varied in any numbers as faras the effect of the present invention can be observed.

Of the deletion, addition, and substitution, the substitution isparticularly preferred in the amino acid variation. Amino acids that aresimilar to each other in hydrophobicity, charge, pK, stereo-structuralcharacteristic, or the like are more preferably replaced with eachother. For example, such substitutable amino acids are in each of thefollowing groups: 1) glycine and alanine; 2) valine, isoleucine, andleucine; 3) aspartic acid, glutamic acid, asparagine, and glutamine; 4)serine and threonine; 5) lysine and arginine; and 6) phenylalanine andtyrosine.

The “osmosensing histidine kinase having no transmembrane region” willbe further explained with the specific examples shown below.

(Osmosensing Histidine Kinase Having No Transmembrane Region ofNeurospora crassa)

A protein OS-1 encoded by an os-1 gene isolated from an osmosensingmutant os-1 of Neurospora crassa can be mentioned as the “osmosensinghistidine kinase having no transmembrane region” (Schumacher, M. M. et.al. (1997) Current Microbiol. 34:340-347, Alex, L. A. et. al. (1996)Proc. Natl. Acad. Sci. USA 93:3416-3421). Amino acid sequences of OS-1and nucleotide sequences of the os-1 gene are published (amino acidsequence: Genebank accession AAB03698, AAB01979, nucleotide sequence:Genebank accession U50263, U53189), and utility of OS-1 and os-1 gene inscreening system for antifungal compounds is described in U.S. Pat. No.5,939,306. Since Neurospora crassa mutant os-1 has the highersensitivity to high osmolarity stress than that of a wild strain, it hasbeen found that OS-1 is an osmosensing histidine kinase involved inosmolarity adaptation in Neurospora crassa. It is known that OS-1 hasthe aforementioned structural characteristic based on its amino acidsequence. In addition, it is known that Neurospora crassa mutant os-1has the resistance to fungicides containing, as an active ingredient, adicarboxyimide antifungal compound, an “aromatic hydrocarbon antifungalcompound” or a phenylpyrrole antifungal compound. Further, a genemutation which leads to an amino acid substitution in a characteristicrepeat sequence region of OS-1 was observed in the os-1 mutant geneisolated from Neurospora crassa mutant exhibiting the resistance to afungicide containing a dicarboxyimide antifungal compound as an activeingredient (Miller, T. K. et. al. (2002) Fungl Gen. Biol. 35:147-155).From the foregoing, it is predicted that an antifungal compoundcontained as an effective ingredient in the aforementioned fungicidetargets OS-1 of Neurospora crassa.

(Osmosensing Histidine Kinase Having No Transmembrane Region ofBotryotinia fuckeliana)

Examples of the “osmosensing histidine kinase having no transmembraneregion” include BcOS-1 of Botryotinia fuckeliana. The BcOS-1 gene wasisolated as a gene homologous to Neurospora crassa OS-1 gene, andnucleotide sequencez and amino acid sequences are published (nucleotidesequence: GeneBank accession AF396287, AF435964, amino acid sequence:GeneBank accession AAL37947, AAL30826). It is known that BcOS-1 has theaforementioned structural characteristic based on its amino acidsequence. In addition, in the BcOS-1 gene isolated from a Botryotiniafuckeliana strain resistant to a fungicide containing a dicarboxyimideantifungal compound as an active ingredient, a mutation which leads toamino acid substitution in the characteristic repeat sequence region ofBcOS-1 was observed, as in the OS-1 gene isolated from a Neurosporacrassa strain resistant to a fungicide containing a dicarboxyimideantifungal compound as an active ingredient. Further, since anantifungal compound-resistant mutant deficient in the BcOS-1 has thehigher osmolarity sensitivity than that of a wild strain, it is knownthat BcOS-1 is osmosensing histidine kinase (Oshima, M. et. al. (2002)Phypotathology 92:75-80).

More specifically, examples of BcOS-1 include BcOS-1 having an aminoacid sequence represented by SEQ ID NO: 1 which was isolated from Bc-16strain described in Example.

(Osmosensing Histidine Kinase Having No Transmembrane Region ofMagnaporthe grisea)

Example of the “osmosensing histidine kinase having no transmembraneregion” include HIK1 of Magnaporthe grisea. The hik1 gene is a genehomologous to Neurospora crass os-1 gene, and a nucleotide sequence andan amino acid sequence are published (nucleotide sequence: Genebankaccession AB041647, amino acid sequence: GeneBank accession BAB40947).It is known that HIK1 has the aforementioned structural characteristicssuch as lack of the transmembrane region based on its amino acidsequence. In addition, it is observed that Magnaporthe grisea deficientin the hik1 gene has the higher osmolarity sensitivity than that of awild strain, demonstrating that HIK1 is an osmosensing histidine kinase(hppt://www.sci.saitama-u.ac.jp/seitai/iden/Japanese/Abst Symp3.html).

More specifically, examples of HIK1 include HIK1 having an amino acidsequence represented by SEQ ID NO: 16 which was isolated from the P-37strain described in Example.

(Definition of Filamentous Fungus and Yeast)

In the present invention, the “filamentous fungus” means fungi otherthan fungi which can be classified as yeast, among fungi consisting ofMyxomycota and Eumycota, described in “Revised Edition, Classificationand Identification of Microorganisms (Volume 1), edited by TakeharuHASEGAWA, Society Publishing Center, 1984 (ISBN 4-7622-7399-6)”.Examples of filamentous fungus classified in Myxomycota includePlasmodiophora brassicae belonging to Plasmodiophoromycetes. Inaddition, examples of filamentous fungus which is classified in Eumycotainclude Phytophthora infestans belonging to Mastigomycotina, Rhizopusstolonifer and Rhizopus oryzae belonging to Zygomycotina, Neurosporacrassa, Mycospharella tritici, Erysiphe graminis, Linocarpon cariceti,Cochliobolus miyabeanus, Botrytinia fuckeliana and Magnaporthe griseabelonging to Ascomycotina, Ustilago maydis, Puccinia recondite andThanatephorus cucumeris belonging to Basidiomycotina, Cladosporiumfulvum, Alternalia kikuchiana and Fusarium oxysporum belonging toDeuteromycotina, and the like.

In addition, yeast means fungi in which they are grown mainly bybudding, a single cell generation is long, a colony formed by growth ofa single cell does not become hairy, but becomes white brightpaste-like” as described in “Revised Edition, Classification andIdentification of Microorganisms (Volume 1), edited by TakeharuHASEGAWA, Society Publishing Center, 1984 (ISBN 4-7622-7399-6)”.Examples thereof include Saccharomyces cerevisiae belonging to genusSaccharomyces, Schizosaccharomyces pombe belonging to genusSchizosaccharomyces, Phichia burtonii belonging to genus Phichia,Candida albicans belonging to genus Candida, and the like.

(Osmosensing Histidine Kinase Having Mutation which Confers Resistanceto any of Dicarboxylmide Antifungal Compound, Aromatic HydrocarbonAntifungal Compound and Phenylpyrrole Antifungal Compound, and Having NoTransmembrane Region)

As a specific example of the “osmosensing histidine kinase having notransmembrane region”, there can also be exemplified “osmosensinghistidine kinase having no transmembrane region” having mutation whichconfers resistance to any of a dicarboxyimide antifungal compound, an“aromatic hydrocarbon antifungal compound” and a phenylpyrroleantifungal compound. Specifically, there can be exemplified BcOS-1having an amino acid sequence represented by SEQ ID NO: 13 which isdescribed in Example.

Herein, the dicarboxyimide antifungal compound is a generic name ofantifungal compounds having dicarboxyimide as a basic structure, andexamples thereof include antifungal compounds described in ModernSelective Fungicide-Properties, Applications, Mechanism of Action-2^(nd)revised and enlarged edition Lyr, H. ed. Gustav Fisher Verlag, New York,USA ISBN 3-334-60455-1 Chapter 6, p 99-118. Specifically, there are acompound having a structure represented by the chemical formula (1)(Procymidone: hereinafter, referred to as Compound (1) in some cases), acompound having a structure represented by the chemical formula (2)(Iprodione: hereinafter, referred to as Compound (2) in some cases), acompound having a structure represented by the chemical formula (3)(Vinclozolin: hereinafter, referred to as Compound (3) in some cases)and the like. The “aromatic hydrocarbon antifungal compound” is ageneric name of antifungal compounds having a benzene ring as a basicstructure, and examples thereof include antifungal compounds describedin Modern Selective Fungicide-Properties, Applications, Mechanism ofAction-2^(nd) revised and enlarged edition Lyr, H. ed. Gustav FisherVerlag, New York, USA ISBN 3-334-60455-1 Chapter 5, p 75-98.Specifically, there are a compound having a structure represented by thechemical formula (4) (Quintozene: hereinafter, referred to as Compound(4) in some cases), a compound having a structure represented by thechemical formula (5) (Tolclofos-methyl: hereinafter, referred to asCompound (5) in some cases). In addition, the phenylpyrrole antifungalcompound is a generic name of antifungal compounds having phenylpyrroleas a basic structure, and examples thereof include antifungal compoundsdescribed in Modern Selective Fungicide-Properties, Applications,Mechanism of Action-2^(nd) revised and enlarged edition Lyr, H. ed.Gustav Fisher Verlag, New York, USA ISBN 3-334-60455-1 Chapter 19, p405-407. Specifically, there are a compound having a structurerepresented by the chemical formula (6) (Fludioxonil: hereinafter,referred to as Compound (6) in some cases), a compound having astructure represented by the chemical formula (7) (Fenpiclonil:hereinafter, referred to as Compound (7) in some cases) and the like.

Chemical formulas of the aforementioned dicarboxyimide antifungalcompound, “aromatic hydrocarbon antifungal compound” and phenylpyrroleantifungal compounds are shown below.

(1) Compound Having a Structure Represented by the Chemical Formula (1)(Compound (1))

(2) Compound Having a Structure Represented by the Chemical Formula (2)(Compound (2))

(3) Compound Having a Structure Represented by the Chemical Formula (3)(Compound (3))

(4) Compound Having a Structure Represented by the Chemical Formula (4)(Compound (4))

(5) Compound Having a Structure Represented by the Chemical Formula (5)(Compound (5))

(6) Compound Having a Structure Represented by the Chemical Formula (6)(Compound (6))

(7) Compound Having a Structure Represented by the Chemical Formula (7)(Compound (7))

The “mutation which confers resistance to any of a dicarboxyimideantifungal compound, an aromatic hydrocarbon antifungal compound and aphenylpyrrole antifungal compound” indicates a mutation which can befound in the “osmosensing histidine kinase having no transmembraneregion” produced by a filamentous fungus mutant having resistance to anyof a dicarboxyimide antifungal compound, an “aromatic hydrocarbonantifungal compound” and a phenylpyrrole antifungal compound, that is,substitution, addition or deletion of one or more amino acids whichconfer resistance to a dicarboxyimide antifungal compound, an “aromatichydrocarbon antifungal compound” and a phenylpyrrole antifungalcompound, provided that mutation by which the “osmosensing histidinekinase having no transmembrane region” becomes not to function ashistidine kinase is eliminated. Herein, a mutant of filamentous fungushaving resistance to any of a dicarboxyimide antifungal compound, an“aromatic hydrocarbon antifungal compound” and a phenylpyrroleantifungal compound may be filamentous fungus isolated from the natureto which any of a dicarboxyimide antifungal compound, an “aromatichydrocarbon antifungal compound” and a phenylpyrrole antifungal compoundwas applied, or may be resistance-acquired filamentous fungus selectedby artificially culturing filamentous fungus in the presence of adicarboxyimide antifungal compound, an “aromatic hydrocarbon antifungalcompound” or phenylpyrrole antifungal compound.

Specifically, in BcOS-1 in the “osmosensing histidine kinase having notransmembrane region” of Botryotinia fuckeliana, amino acid-substitutionI365S which confers resistance to a dicarboxyimide antifungal compoundis reported in Oshima, M. et al. (2002) Phytopathology 92:75-80 (herein,“I365S” means that isoleucine at amino acid residue 365 is substitutedwith serine. Hereinafter, amino acid substitution is describedsimilarly). As an amino acid substitution which confers resistance to adicarboxyimide antifungal compound in OS-1 which is the “osmosensinghistidine kinase having no transmembrane region” of Neuorspora crassa,T368P, Q388S, E418E, L459M, A578V, G580R, 1582M, M639V, A578V, G580G andL625P are reported and, as an amino acid deletion, 680K is reported inMiller, T. K. et al. (2002) Fungal Gen. Biol. 35:147-155 (hereinafter,680K means that lysine at amino acid residue 680 is deleted.Hereinafter, amino acid deletion is described similarly). In addition,amino acid substitution which confers resistance to a phenylpyrroleantifungal compound in the OS-1 of Neurospora crassa, A578V, G580R andL625P are reported in Ochiai, N. et al. (2001) Pest Management Sci.57:437-442.

Besides the aforementioned resistance mutation, resistance mutation maybe found by analyzing an amino acid sequence of the “osmosensinghystidine kinase having no transmembrane region” isolated from a mutantfilamentous fungus having resistance to any of a dicarboxyimideantifungal compound, an “aromatic hydrocarbon antifungal compound” and aphenypyrrole antifungal compound, and comparing with an amino acidsequence of the protein in a sensitive wild strain.

(Preparation of Transformed Cell in which a Polynucleotide Having aNucleotide Sequence Encoding an Amino Acid Sequence of OsmosensingHistidine Kinase Having No Transmembrane Region is Introduced in aFunctional Form into a Cell Deficient in at Least One Hybrid-SensorKinase)

The transformed cell in which a polynucleotide having a nucleotidesequence encoding an amino acid sequence of osmosensing histidine kinasehaving no transmembrane region (hereinafter, referred to as presenthistidine kinase in some cases) is introduced in functional form, can beobtained by introducing a “polynucleotide having a nucleotide sequenceencoding an amino acid sequence of the present hystidine kinase” or thelike into a “cell deficient in at least one hybrid-sensor kinase” whichis to be a host cell, as described below.

Examples of the “polynucleotide having a nucleotide sequence encoding anamino acid sequence of the present hystidine kinase” include apolynucleotide having a nucleotide sequence encoding an amino acidsequence of the present hystidine kinase which is derived from aplant-pathogenic filamentous fungus, more specifically, for example, apolynucleotide having a nucleotide sequence encoding an amino acidsequence selected from the group consisting of:

(a) an amino acid sequence of an osmosensing histidine kinase having notransmembrane region, which has an amino acid sequence homology of 95%or more to the amino acid sequence represented by any of SEQ ID NOs: 41,55 and 68;

(b) an amino acid sequence of an osmosensing histidine kinase having notransmembrane region, which is encoded by a DNA amplified by apolymerase chain reaction using a Fusarium oxysporum-derived cDNA as atemplate and using an oligonucleotide having the nucleotide sequencerepresented by SEQ ID NO: 52 and an oligonucleotide having thenucleotide sequence represented by SEQ ID NO: 53 as primers;

(c) an amino acid sequence of an osmosensing histidine kinase having notransmembrane region, which is encoded by a DNA amplified by apolymerase chain reaction using a Mycospharella tritici-derived cDNA asa template and using an oligonucleotide having the nucleotide sequencerepresented by SEQ ID NO: 64 and an oligonucleotide having thenucleotide sequence represented by SEQ ID NO: 65 as primers;

(d) an amino acid sequence of an osmosensing histidine kinase having notransmembrane region, which is encoded by a DNA amplified by apolymerase chain reaction using a Thanaphthorus cucumeris-derived cDNAas a template and using an oligonucleotide having the nucleotidesequence represented by SEQ ID NO: 85 and an oligonucleotide having thenucleotide sequence represented by SEQ ID NO: 86 as primers;

(e) an amino acid sequence of an osmosensing histidine kinase having notransmembrane region, which is derived from Phytophthora infestans andhas the amino acid sequence represented by SEQ ID NO: 90;

(f) the amino acid sequence represented by SEQ ID NO: 41;

(g) the amino acid sequence represented by SEQ ID NO: 55; and

(h) the amino acid sequence represented by SEQ ID NO: 68.

One example of a process for producing the transformed cell will beshown below.

(1) Preparation of cDNA

First, total RNA is prepared from filamentous fungus, for example,according to the method described in Molecular Cloning 2nd editionauthored by J., Sambrook, E., F., Frisch, T., Maniatis. Specifically,for example, a part of a fungal tissue is collected from Neurosporacrassa, Botrytinia fuckeliana, Magnaporthe grisea, Phytophthorainfestans, Thanatephorus cucumeris, Fusarium oxysporum, Mycospharellatritici, Thanatephorus cucumeris, Thanatephorus cucumeris and the like,the collected tissue is frozen in liquid nitrogen, and is physicallyground with a mortar or the like. Then, total RNA may be prepared by theconventional method such as (a) a method of adding a solution containingguanidine hydrochloride and phenol or a solution containing SDS andphenol to the resulting ground material, to obtain total RNA, or (b) amethod of adding a solution containing guanidine thiocyanate to theaforementioned ground material, and further adding CsCl, followed bycentrifugation, to obtain total RNA. In the procedures, a commerciallyavailable kit such as RNeasy Plant Mini Kit (manufactured by QIAGEN) maybe also used.

Then, the thus prepared total RNA is used to prepare a cDNA. Forexample, cDNA may be prepared by reacting a reverse transcriptase on thetotal RNA after an oligo-dT chain or a random primer is annealed tototal RNA. In addition, further, a double-stranded cDNA can be prepared,for example, by reacting RNaseH, DNA Polymerase I on said cDNA. In theprocedures, a commercially available kit such as SMART™ PCR cDNASynthesis Kit (manufactured by Clonech), cDNA Synthesis Kit(manufactured by TAKARA SHUZO Co., Ltd.), cDNA Synthesis Kit(manufactured by Amersham Pharmacia) and ZAP-cDNA Synthesis Kit(manufactured by Stratagene) can be used.

(2) Cloning

When a nucleotide sequence of a desired present histidine kinase isknown, a polynucleotide having a nucleotide sequence encoding an aminoacid sequence of the present histidine kinase can be obtained, forexample, from the cDNA prepared as described above, for example, by PCRusing as a primer an oligonucleotide having a partial nucleotidesequence of the known nucleotide sequence, or a hybridization methodusing as a probe an oligonucleotide having a partial nucleotide sequenceof the known nucleotide sequence.

A polynucleotide having a nucleotide sequence encoding an amino acidsequence of BcOS-1 which is the present histidin kinase can be preparedfrom a cDNA of Botryotinia fuckeliana, for example, by PCR using as aprimer an oligonucleotide having a partial nucleotide sequence of thenucleotide sequence represented by SEQ ID NO: 2, or a hybridizationmethod using as a probe an oligonucleotide having a partial nucleotidesequence of the nucleotide sequence represented by SEQ ID NO: 2.

In addition, a polynucleotide having a nucleotide sequence encoding anamino acid sequence of HIK1 which is the present histidine kinase can beobtained from a cDNA of Magnaporthe grisea, for example, by PCR using asa primer an oligonucleotide having a partial nucleotide sequence of thenucleotide sequence represented by SEQ ID NO: 17, or hybridizationmethod using as a probe an oligonucleotide having a partial nucleotidesequence of the nucleotide sequence represented by SEQ ID NO: 17.

When a nucleotide sequence of a desired present histidine kinase isunknown, a polynucleotide having a nucleotide sequence encoding an aminoacid sequence of the present histidine kinase can be obtained by ahybridization method using as a probe an oligonucleotide having apartial nucleotide sequence of the nucleotide sequence of the presenthistidine kinase, the nucleotide sequence of which is known, or by PCRusing as a primer an oligonucleotide designed based on a highlyhomologous amino acid sequence in plural present histidine kinases, anamino acid sequence of which is known. As the highly homologous aminoacid sequence among plural present histidine kinases, amino acidsequences of which are known, for example, there can be exemplifiedamino acid sequences of a conserved motifs observed in the “repeatsequence region”, the “histidine kinase region”, the “receiver region”and the like, characterized in the structure of the present histidinekinase.

More specifically, when the BcOS-1 gene of Botryotinia fuckeliana isobtained by PCR, for example, oligonucleotides designed and synthesizedbased on nucleotide sequences of about 20 bp to about 40 bp which areselected from a 5′ non-translated region and a 3′ non-translated region,respectively, of the nucleotide sequence represented by SEQ ID NO: 2 canbe used as a primer set. Examples of the primer set include a set of anoligonucleotide consisting of the nucleotide sequence represented by SEQID NO: 3 and an oligonucleotide consisting of the nucleotide sequencerepresented by SEQ ID NO: 4. A PCR reaction solution to be used may beprepared by adding a reaction solution designated by a commerciallyavailable DNA polymerase or kit as described below to 250 ng of a cDNA.The PCR reaction conditions can be appropriately changed depending on aprimer set to be used, and examples thereof include the condition ofmaintaining a temperature at 94° C. for 2 minutes, then maintaining atemperature at about 8° C. for 3 minutes and, thereafter, repeatingaround 40 cycles of incubation, each cycle comprising maintaining atemperature at 94° C. for 30 seconds, then at 55° C. for 30 seconds,then at 72° C. for 4 minutes, and the condition of repeating 5 to 10cycles of incubation, each cycle comprising maintaining a temperature at94° C. for 5 seconds, then at 72° C. for 4 minutes, and furtherrepeating about 20 to 40 cycles of incubation, each cycle comprisingmaintaining a temperature at 94° C. for 5 seconds, then at 70° C. for 4minutes. For the procedures, commercially available DNA polymerasescontained in Heraculase™ Enhanced DNA Polymerase (manufactured by ToyoboCo., Ltd.), Advantage cDNA PCR Kit (manufactured by Clonetech), andcommercially available kits such as TAKARA Ex Taq (manufactured byTAKARA SHUZO Co., Ltd.), PLATINUM™ PCR SUPER Mix (manufactured byLifetech Oriental), KOD-Plus- (manufactured by Toyobo Co., Ltd.) and thelike can be used.

When the hik1 gene of Magnaporthe grisea is obtained by PCR, forexample, oligonucleotides designed and synthesized based on nucleotidesequences selected from a 5′ non-translation region and a 3′non-translation region, respectively, of the nucleotide sequencerepresented by SEQ ID NO: 17 can be used as a primer set. Examples ofthe primer set include a set of an oligonucleotide comprising thenucleotide sequence represented by SEQ ID NO: 18 and an oligonucleotidecomprising the nucleotide sequence represented by SEQ ID NO: 19. A PCRreaction solution and the reaction conditions as described above can beused to perform PCR, to obtain the hik1 gene.

When a gene of the present histidine kinase, a nucleotide sequence ofwhich is not known, is obtained from Fusarium oxysporum, Mycospharellatritici, Thanatephorus cucumeris or Phytophthora infestans, apolynucleotide having a nucleotide sequence encoding a part of an aminoacid sequence of the present histidine kinase (hereinafter, referred toas present gene fragment in some cases) can be obtained by the followingPCR. As a primer set, for example, a set of oligonucleotides designedand synthesized based on amino acid sequences of a conserved motifsobserved in the “repeat sequence region”, the “histidine kinase region”,the “receiver region” and the like, characterized in the structure ofthe present histidine kinase, can be used. Examples of the primer setinclude a primer set of an oligonucleotide having the nucleotidesequence represented by any of SEQ ID NOs: 30 to 34 and anoligonucleotide having the nucleotide sequence represented by any of SEQID NOs: 35 to 40.

Specifically, in the case of Fusarium oxysporum, for example, using anoligonucleotide primer having the nucleotide sequence represented by SEQID NO: 33 and an oligonucleotide primer having the nucleotide sequencerepresented by SEQ ID NO: 38, and using KOD-Plus- (TOYOBO),amplification is performed under the conditions in which a temperatureis maintained at 94° C. for 2 minutes, and then 35 cycles of incubationare repeated, each cycle comprising maintaining a temperature at 94° C.for 15 seconds, then, at 55° C. for 30 seconds and, further, at 68° C.for 5 minutes. In addition, in the case of Mycospharella tritici, forexample, using an oligonucleotide primer having the nucleotide sequencerepresented by SEQ ID NO: 31 and an oligonucleotide primer having thenucleotide sequence represented by SEQ ID NO: 40, and using KOD-Plus-(TOYOBO), amplification is performed under the conditions in which atemperature is maintained at 94° C. for 2 minutes, and 35 cycles ofincubation are repeated, each cycle comprising maintaining a temperatureat 94° C. for 15 seconds, then, at 55° C. for 30 seconds and, further,at 68° C. for 3 minutes. In addition, in the case of Thanatephoruscucumeris, for example, using an oligonucleotide primer having thenucleotide sequence represented by SEQ ID NO: 30 and an oligonucleotideprimer having the nucleotide sequence represented by SEQ ID NO: 37, andusing KOD-Plus- (TOYOBO), amplification is performed under theconditions in which a temperature is maintained at 94° C. for 2 minutes,and 35 cycles of incubation are repeated, each cycle comprisingmaintaining a temperature at 94° C. for 15 seconds, then, at 55° C. for30 seconds, further, at 68° C. for 1 minute. In addition, in the case ofPhytophthora infestans, for example, using an oligonucleotide primerhaving the nucleotide sequence represented by SEQ ID NO: 31 and anoligonucleotide primer having the nucleotide sequence represented by SEQID NO: 37, and using KOD-Plus- (TOYOBO), amplification is performedunder the conditions in which a temperature is maintained at 94° C. for2 minutes, and 35 cycles of incubation are repeated, each cyclecomprising maintaining a temperature at 94° C. for 15 seconds, then, at55° C. for 30 seconds and, further, at 68° C. for 1 minute. By such thePCR, a polynucleotide having a nucleotide sequence encoding a part of anamino acid sequence of the present hisitidine kinase is amplified. Apolynucleotide having a nucleotide sequence encoding a full length aminoacid sequence of the present histidine kinase can be obtained by RACEmethod by using, for example, SMART RACE cDNA Amplification Kit(CLONTECH) and primers designed based on a nucleotide sequence of theamplified polynucleotide (present gene fragment).

When the polynucleotide obtained as described above has revealed anucleotide sequence encoding a full length amino acid sequence of thepresent histidine kinase, by PCR using an oligonucleotide having apartial nucleotide sequence of the sequence as a primer, apolynucleotide having a nucleotide sequence encoding an amino acidsequence of the present histidine kinase may be also obtained.

Specifically, when a gene of the present histidine kinase of Fusariumoxysporum (hereinafter, referred to FoOS-1 gene in some cases) isobtained by PCR, for example, oligonucleotides designed and synthesizedbased on nucleotide sequences selected from a 5′-terminal region and a3′-terminal region, respectively, of the nucleotide sequence representedby SEQ ID NO: 42 can be used as a primer set. Examples of the primer setinclude a set of an oligonucleotide comprising the nucleotide sequencerepresented by SEQ ID NO: 52 and an oligonucleotide comprising thenucleotide sequence represented by SEQ ID NO: 53. A PCR reactionsolution and the reaction conditions as described above are used toperform PCR, whereby, a polynucleotide having a nucleotide sequenceencoding an amino acid sequence of the present histidine kinase derivedfrom Fusarium oxysporum can be obtained.

In addition, when a gene of the present histidine kinase ofMycospharella tritici (hereinafter, referred to StOS-1 gene in somecases) is obtained by PCR, for example, oligonucleotides designed andsynthesized based on nucleotide sequences selected from a 5′-teminalregion and a 3′-terminal region, repectively, of the nucleotide sequencerepresented by SEQ ID NO: 56 can be used as a primer set. Examples ofthe primer set include a set of an oligonuceleotide comprising thenucleotide sequence represented by SEQ ID NO. 64 and an oligonucleotidecomprising the nucleotide sequence represented by SEQ ID NO: 65. A PCRreaction solution and the reaction conditions as described above areused to perform PCR, whereby, a polynucleotide having a nucleotidesequence encoding an amino acid sequence of the present histidine kinasederived from Mycospharella tritici can be obtained.

In addition, when a gene of the present histidine kinase ofThanatephorus cucumeris (hereinafter, referred to RsOS-1 gene in somecases) is obtained by PCR, for example, oligonucleotides designed andsynthesized based on nucleotide sequences selected from a 5′-terminalregion and a 3′-terminal region, respectively, of the nucleotidesequence represented by SEQ ID NO: 69 can be used as a primer set.Examples of the primer set include a set of an oligonucleotidecomprising the nucleotide sequence represented by SEQ ID NO: 85 and anoligonucleotide comprising the nucleotide sequence represented by SEQ IDNO: 86. A PCR reaction solution and the reaction conditions as describedabove are used to perform PCR, whereby, a polynucleotide having anucleotide sequence encoding an amino acid sequence of the presenthistidine kinase derived from Thanatephorus cucumeris can be obtained.

When a hybridization method is used, cloning can be performed, forexample, according to the method described in Molecular Cloning 2ndedition, authored by J., Sambrook, E., F., Frisch, T., Maniatis.

A probe used to obtain a gene of the present histidine kinase can beobtained by synthesizing a DNA (around about 200 bases to about 500bases in length) having a partial nucleotide sequence of the nucleotidesequence represented by SEQ ID NO: 2, followed by radioisotope-labelingor fluorescently labeling the DNA according to the conventional method.In such the labeling of a DNA, commercially available kits such asRandom Primed DNA Labelling Kit (manufactured by Boehringer), RandomPrimer DNA Labelling Kit Ver. 2 (manufactured by TAKARA SHUZO Co.,Ltd.), ECL Direct Nucleic acid Labelling and Detection System(manufactured by Amersham Pharmacia), Megaprime DNA-labelling system(manufactured by Amersham Pharmacia) and the like may be utilized. Thethus obtained probe can be used for cloning a gene of the histidinekinase such as the BcOS1-gene of Botrytinia fuckeliana, a nucleotidesequence of which is known, or a gene of the present histidine kinase, anucleotide sequence of which is unknown.

Examples of the hybridization condition include the stringent condition,specifically, the condition under which, in the presence of 6×SSC (0.9 MNaCl, 0.09 M trisodium citrate), 5×Denhart's solution (0.1% (w/v) Ficoll400, 0.1% (w/v) polyvinylpyrrolidone, 0.1% BSA), 0.5% (w/v) SDS and 100μg/ml denatured salmon sperm DNA, or in DIG EASY Hyb solution(Boehringer Manheim) containing 100 μg/ml denatured salmon sperm DNA, atemperature is maintained at 65° C., then a temperature is maintained atroom temperature for 15 minutes twice in the presence of 1×SSC (0.15 MNaCl, 0.015 M trisodium citrate) and 0.5% SDS, further, a temperature ismaintained at 68° C. for 30 minutes in the presence of 0.1×SSC (0.015 MNaCl, 0.0015M trisodium citrate) and 0.5% SDS.

Specifically, for example, for obtaining the BcOS-1 gene of Botrytiniafuckeliana, PCR is performed by using a Botrytinia fuckeliana cDNAlibrary phage solution (about 1,000,000 pfu) as a template, and usingTAKARA LA Taq™ (manufactured by TAKARA SHUZO Co., Ltd.), and using anoligonucleotide comprising the nucleotide sequence represented by SEQ IDNO: 9 and an oligonucleotide comprising a nucleotide sequencecomplementary to the nucleotide sequence represented by SEQ ID NO: 10 asa primer set, whereby, a DNA for a probe is amplified, which may becollected. A PCR reaction solution to be used may be prepared by addinga reaction solution designated by a kit as described above to 250 ng ofa DNA library. Examples of the PCR reaction condition include thecondition under which amplification is performed by maintaining atemperature at 94° C. for 2 minutes, then at 8° C. for 3 minutes, andrepeating 40 cycles of incubation, each cycle comprising maintaining atemperature at 94° C. for 30 seconds, then, at 55° C. for 30 secondsand, then, at 68° C. for 5 minutes. Then, a probe labeled with ³²P canbe prepared by using the amplified and obtained DNA as a template, andusing Megaprime DNA-labelling system (Amersham Pharmacia) and using areaction solution designated by the kit. The thus prepared probe is usedto perform colony hybridization according to the conventional method, inwhich a temperature is maintained at 65° C. in the presence of 6×SSC(0.9 M NaCl, 0.09M trisodium citrate, 5×Denharp's solution (0.1% (w/v)Ficoll 400, 0.1% (w/v) polyvinylpyrrolidone, 0.1% BSA), 0.5% (w/v) SDSand 100 μg/ml denatured Salmon sperm DNA, or in DIG EASY Hyb solution(Boehringer Mannheim), containing 100 μg/ml denated Salmon sperm DNA,then, a temperature is maintained at room temperature for 15 minutestwice in the presence of 1×SSC (0.15 M NaCl, 0.015M trisodium citrate)and 0.5% SDS and, further, a temperature is maintained at 68° C. for 30minutes in the presence of 0.1×SSC (0.015 M NaCl, 0.0015 M sodiumcitrate) and 0.5% SDS, whereby, a clone which hybridizes with the probecan be obtained.

In addition, a gene of the present histidine kinase having a knownnucleotide sequence may be also prepared by performing chemicalsynthesis of a nucleic acid, for example, according to the conventionalmethod such as a phosphite triester method (Hunkapiller, M. et al,Nature 310, 105, 1984), based on the known nucleotide sequence.

The thus obtained polynucleotide having a nucleotide sequence encodingan amino acid sequence of the present histidine kinase may be clonedinto a vector according to the conventional method described in“Molecular Cloning: A Laboratory Manual 2nd edition” (1989), Cold SpringHarbor Laboratory Press, “Current Protocols In Molecular Biology”(1987), John Wiley & Sons, Inc. ISBNO-471-50338-X or the like. Examplesof the vector to be used include pBlueScript II vector (manufactured byStratagene), pUC18/19 vector (manufactured by TAKARA SHUZO Co., Ltd.),TA Cloning vector (manufactured by Invitrogen) and the like.

A nucleotide sequence of the cloned gene may be confirmed by the MaxamGilbert method (described in Maxam, A. M. & W. Gilbert, Proc. Natl.Acad. Sci. USA, 74, 560, 1977 etc.) or the Sanger method (described inSanger, F.& A. R. Coulson, J. Mol. Biol., 94, 441, 1975, Sanger, F, &Nicklen and A. R. Coulson., Proc. Natl. Acad. Sci. USA, 74, 5463, 1977etc.). For the procedures, commercially available kits such as TermoSeqenase II dye terminator cycle sequencing kit (manufactured byAmersham Pharmacia), Dye Terminator Cycle Sequencing FS Ready ReactionKit (manufactured by PE Biosystems Japan) and the like can be used.

(3) Construction of Expression Vector

An expression vector of a polynucleotide having a nucleotide sequenceencoding an amino acid sequence of the present histidine kinase may beconstructed by a conventional method (for example, method described inJ. Sambrook, E., F., Frisch, T., Maniatis, Molecular Cloning 2ndedition, published by Cold Spring Harbor Laboratory Press etc.).

For example, A polynucleotide having a nucleotide sequence encoding anamino acid sequence of the present histidine kinase may be incorporatedinto a vector which can be utilized in a host cell to be transformed,for example, a vector which contains genetic information required to bereplicable in a host cell, can replicates autonomously, can be isolatedand purified from a host cell, and has a detectable marker (hereinafterreferred to as basic vector in some cases). As the basic vector,specifically, when a bacterium such as Escherichia coli is used as ahost cell, for the example, a plasmid pUC119 (manufacture d by TAKARASHUZO Co., Ltd.), phagemid pBluescriptII (manufactured by Stratagene)and the like may be used. When yeast is used as a host cell, forexample, plasmids pACT2 (manufactured by Clontech), p415 CYC(ATCC87382), p415 ADH (ATCC87374) and the like may be used. When a plantcell is used as a host cell, for the example, a plasmid pBI221(Clontech) and the like may be used.

An expression vector which can express a polynucleotide having anucleotide sequence encoding an amino acid sequence of the presenthistidine kinase in a host cell can be constructed by incorporating intoa basic vector a polynucleotide having a nucleotide sequence encoding anamino acid sequence of the present histidine kinase upstream of which apromoter functional in a host cell is operably linked. Herein, the“operably linked” means that the promoter and a polynucleotide having anucleotide sequence encoding an amino acid sequence of the presenthistidine kanase are ligated so that the polynucleotide having anucleotide sequence encoding an amino acid sequence of the presenthistidine kinase is expressed under control of the promoter in a hostcell. Examples of a promoter functional in a host cell include, when ahost cell is Escherichia coli, a promoter of a lactose operon (lacP) apromoter of tryptphan operon (trpP), a promoter of an arginine operon(argP), a promoter of a galactose operon (galP), tac promoter, T7promoter, T3 promoter of Escherichia coli, a promoter of λ phage (λ-pL,λ-pR) and the like. In addition, when a host cell is yeast, examplesinclude an ADH1 promoter, a CYC1 promoter and the like. The ADH1promoter can be prepared, for example, by the conventional geneticengineering method from a yeast expression vector p415 ADH (ATCC87374)harboring an ADH1 promoter and a CYC1 terminator. The CYC1 promoter canbe prepared by the conventional genetic engineering method from p415CYC(ATCC87382). Examples of the promoter include, when a host cell is aplant cell, a nopaline synthase gene (NOS) promoter, an octopinesynthase gene (OCT) promoter, a cauliflower mosaic virus (CaMV)-derived19S promoter, a CaMV-derived 35S promoter and the like.

In addition, when a polynucleotide having a nucleotide sequence encodingan amino acid sequence of the present histidine kinase is incorporatedinto a vector already harboring a promoter functional in a host cell, agene of the present histidine kinase may be inserted into downstream ofthe promoter so that a promoter harbored by the vector and a gene of thepresent histidine kinase are operably linked. For the example, theaforementioned yeast plasmid p415 ADH has an ADH1 promoter and, when agene of the present histidine kinase is inserted downstream of an ADH1promoter of the plasmid, an expression vector which can express a geneof the present histidine kinase in a budding yeast such as Saccharomycescerevisiae AH22 (IFO10144) and TM182 (Maeda, T. et al. (1994) Nature369:242-245) can be constructed.

(4) Preparation of Transformed Cell

By introducing the constructed expression vector into a host cellaccording to the conventional method, a transformed cell expressing thepresent histidine kinase can be prepared. As a host cell used forpreparing such the transformed cell, for example, there are bacterium,yeast, plant cell and the like. As the bacterium, for example, there areEscherichia coli, Vibrio harveiy and the like. As the yeast, there arebudding yeast and diving yeast. More specifically, for example, thereare yeasts belonging to genus Saccharomyces, genus Shizosaccharomycessthe like. As a plant cell, for example, there is a plant cell such asArabidopsis thaliana and the like.

As a method of introducing an expression vector into the aforementionedhost cell, the conventional introducing method can be applied dependingon a host cell to be transformed. For example, when bacterium is used asa host cell, the expression vector can be introduced into a host cell bythe conventional introducing method such as a calcium chloride methodand an electroporation method described in Moleculer Cloning (J.Sambrook et al., Cold spring Harbor, 1989). When yeast is used as a hostcell, for example, the expression vector can be introduced into a hostcell using Yeast transformation kit (Clontech) based on a lithiummethod. In addition, when a plant cell is used as a host cell, forexample, the expression vector can be introduced into a host cell usingthe conventional introducing method such as an Agrobacterium infectionmethod (JP-B No. 2-58917 and JP-A No. 60-70080), an electroporationmethod into a propoplast (JP-A No. 60-251887 and JP-A No. 5-68575) and aparticle gun method (JP-A No. 5-508316 and JP-A No. 63-258525).

(Intracellular Signal Transduction System Regarding Present HistidineKinase)

In the present invention, in order to measure an amount of intracellularsignal transduction from the present histidine kinase expressed in thetransformed cell prepared as described above or an index value havingthe correlation therewith, an intracellular signal transduction systemoriginally contained in a host cell used for preparing the transformedcell may be utilized. Examples of the intracellular signal transductionsystem which can be utilized include an intracellular signaltransduction system regarding osmolarity responses of the aforementionedbudding yeast, an intracellular signal transduction system regardingcell cycle progression and oxidative stress response of fission yeast,an intracellular signal transduction system regarding control ofexpression of capsular polysaccharide biosynthesis operon in Escherichiacoli, an intracellular signal transduction system regarding control ofcell density-sensitive luminescence of bioluminescent marinemicroorganism Vibrio harveyi, an intracellular signal transductionsystem regarding cytokinin response of Arabidopsis thaliana and thelike.

When the aforementioned expression vector of the present histidinekinase is introduced using the “cell deficient in at least onehybrid-sensor kinase” as a host cell used for preparing such thetransformed cell, the produced present histidine kinase functions inplace of deleted hybrid-sensor kinase, and intracellular signal istransmitted. In the case where a test substance is contacted with thetransformed cell, when signal transduction from the present histidinekinase is inhibited by the test substance, change in an amount of growthof the transformed cell, change in morphology of the transformed cell,change in a shape of the transformed cell, change in an amount ofbiosynthesis of a particular substance in the cell, change in an amountof metabolism of a particular substance in the cell and the like occurin some cases. In such the cases, an antifungal activity of the testsubstance acting on the present histidine kinase can be measured usingchange in an amount of growth of the transformed cell, change inmorphology, change in shape, change in an amount of biosynthesis of aparticular substance in a cell, change in an amount of metabolism of aparticular substance and the like as an index.

On the other hand, when at least one intrinsic hybrid-sensor kinase isnot deleted in a host cell used for preparing a transformed cell, thereare both of signal transduction from intrinsic hybrid-sensor kinases andintracellular signal transduction from the introduced present histidinekinase in intracellular signal transduction of the transformed cell.Change in an amount of growth of the transformed cell, change inmorphology, change in shape, change in am amount of biosynthesis of aparticular substance in the cell, change in an amount in metabolism of aparticular substance in the cell and the like reflecting an amount ofintracellular signal transduction from the introduced present histidinekinase become smaller by the influence of an amount of intracellularsignal transduction from intrinsic hybrid-sensor kinase. In the presentinvention, by using a host cell deficient in at least one intrinsichybrid-sensor kinase, since change in an amount of growth of thetransformed cell, change in morphology, change in shape, change in anamount of biosynthesis of a particular substance in the cell, change inan amount of metabolism of particular substance in the cell and the likereflecting an amount of intracellular signal transduction from theintroduced present histidine kinase become larger, the sensitivity ofthe transformed cell to an antifungal compound is enhanced. Like this,the transformed cell with the enhanced sensitivity to an antifungalcompound is useful for assaying the antifungal activity of a testsubstance and searching an antifungal compound using the assay.

Specifically, when the present histidine kinase is introduced in aSaccharomyces cerevisiae strain deficient in hybrid-sensor kinase SLN1(Maeda, T. et al. Nature: 369 242-245 (1994)), the present histidinekinase performs signal transduction in place of deficient SLN1, whereby,an amount of intracellular signal transduction from the introducedpresent histidine kinase can be detected more clearly using an amount ofgrowth of host cell as an index. That is, when the test substance actson the present histidine kinase, and an amount of signal transductionfrom the present histidine kinase in a host cell is changed, it can beclearly measured as change in an amount of growth of the transformedbudding yeast. In addition, an Escherichia coli strain deficient in ahybrid-sensor kinase RcsC, a fission yeast strain deficient in PHK1 toPHK3 involved in control of cell cycle progression, a Vibrio harveyistrain deficient in LuxN associated with control of celldensity-sensitive luminescence and an Arabidopsis thaliana straindeficient in cytokinin receptor CRE1 can be exemplified as onepreferable aspect of the “cell deficient in at least one hybrid-sensorkinase”.

(Method of Assaying Antifungal Activity of Test Substance)

In a method of assaying the antifungal activity of a test substance, anembodiment of a first step of culturing a transformed cell in which apolynucleotide having a nucleotide sequence encoding an amino acidsequence of the present histidine kinase introduced in the presence of atest substance includes a method of contacting a test substance with thetransformed cell by culturing the transformed cell in a mediumcontaining the test substance. Culturing the transformed cell may be anyform of liquid culturing in which the cell is cultured in a liquidmedium, solid culturing in which the cell is cultured on a solid mediumprepared by adding agar or the like to liquid medium, and the like. Theconcentration of a test substance in the medium is, for example, about 1nm to about 1 mM, preferably about 10 nm to about 100 μM. A culturingtime is, for example, about 1 hour or longer and around 3 days,preferably about 25 hours to around 2 days. When the antifungal activityof a test substance is assayed, as a medium containing a test substance,an antifungal compound-free medium may be used.

An amount of intracellular signal transduction from the presenthistidine kinase expressed in a transformed cell cultured in the firststep or an index value having the correlation therewith is measured.And, the antifungal activity of a test substance is assayed based on adifference between an amount of intracellular signal transduction or anindex value having the correlation therewith measured in the second stepand a control. For example, the antifungal activity of the testsubstance can be assessed based on a difference obtained by comparingamounts of intracellular signal transduction or index values having thecorrelation therewith, which are measured as described above in sectionsin which different two or more substances (for example, it is preferablethat among different two or more substances, at least one substance hasno antifungal activity) are independently used, respectively, as a testsubstance.

Specifically, for example, when a transformed cell prepared by using, asa host cell, the TM182 (SLN1A) strain (Maeda T. et al. Nature: 369242-245 (1994)) which is a SLN1 gene-deficient strain in which the PTP2Tyrosine phosphatase gene (Ota et al, Proc. N. A. sic. USA, 89,2355-2359 (1992)) introduced (that is, a transformed cell having thefunction that cell growth is directly controlled by transduction of anintracellular signal from the present histidine kinase) is used, theantifungal activity can be measured by using, as an index, an amount ofgrowth of the transformed cell in a medium (agar medium or liquidmedium) using glucose as a carbon source, for example, Glu-Ura-Leumedium. When a medium in which a test substance is added to theGlu-Ura-Leu medium (medium containing no antifungal compound) is used, atest substance inhibiting growth of the transformed cell can be assessedto have the antifungal activity. In addition, as a control, it is enoughto examine that growth of the transformed cell in a medium usinggalactose in place of glucose as a carbon source, for example,Gal-Ura-Leu medium is observed regardless of the presence or the absenceof test substance.

When a transformed cell prepared by using, as a host cell, fission yeastwhich is PHK1, PHK2 and PHK3 gene-deficient strain (that is, atransformed cell in which cell cycle progression is directly regulatedby transduction of an intracellular signal from the histidine kinase) isused, cell diviision of the fission yeast may be observed under amicroscope. When a medium in which a test substance is added to a mediumcontaining no substance having the antifungal activity is used, a testsubstance which shortens a cell length of a dividing cell of thetransformed cell can be assessed to have the antifungal activity.

When a transformed cell prepared by using, as a host cell, RcsCgene-deficient Escherichia coli in which cps-LacZ introduced is used,color development of X-Gal may be observed in an agar medium or a liquidmedium (Suzuki et al. Plant Cell Physiol. 42:107-113 (2001)). When amedium in which a test substance is added to a medium containing nosubstance having the antifungal activity is used, a test substance whichcan make the transformed cell develop blue can be assessed to have theantifungal activity.

In addition, when a transformed cell prepared by using, as a host cell,LuxN gene-deficient V. harveyi (i.e. a transformed cell in whichbioluminescence is directly regulated by transduction of anintracellular signal from the present histidine kinase) is used, thefluorescent light emitted by the transformed microorganism may beobserved. When a medium containing a test substance and not containing asubstance having the antifungal activity is used, a test substance whichmake the transformed cell possible to emit the fluorescent light can beassessed to have the antifungal activity.

Further, a substance having the antifungal activity can be also searchedby selecting an antifungal compound based on the antifungal activityassessed by the aforementioned assaying method.

Effects of the Invention

The present invention can provide a transformed cell with the enhancedsensitivity to an antifungal compound, a method of assaying theantifungal activity of a test substance using the transformed cell, anda method of searching an antifungal compound using the method.

EXAMPLES

The present invention is further described in the following Examples,which are not intended to restrict the invention.

Example 1 Isolation of Botryotinia fuckeliana BcOS-1 Gene

Total RNA was prepared from Botryotinia fuckeliana. 100 mg of a hypha ofBotryotinia fuckeliana strain Bc-16 grown on a potato dextrose agarmedium (PDA medium manufactured by NISSUI Pharmaceutical Co., Ltd.) wasscratched off, and this was ground in liquid nitrogen using a mortar anda pestle. A RNA was prepared from frozen ground powder using RNeasyPlant Mini Kit (QIAGEN). A frozen ground powder together with liquidnitrogen was transferred to a 50 ml sample tube and, after liquidnitrogen was all volatilized, a solution obtained by adding 10 μL ofmercaptoethanol per 1 ml of a buffer RLC attached to kit was added,followed by stirring. Further, ground powder was well dispersed by a fewof pipettings, and was incubated at 56° C. for 3 minutes. Thereafter,the solution containing ground powder was supplied to QIAshredder spincolumn attached to the kit, and centrifuged at 8,000×g for 2 minutes.The filtration supernatant was transferred to a fresh sample tube, a0.5-fold volume of 99.5% ethanol was added thereto, and the material waswell mixed by pipetting. This mixture was supplied to RNeasy mini spincolumn attached to the kit, and centrifuged at 8,000×g for 1 minute. Thefiltrate was discarded, the residue was added 700 μL of a buffer RW1attached to the kit, and centrifuged at 8,000×g for 1 minute, and thefiltrate was discarded. Further, the residue was added 500 μL of abuffer RPE attached to the kit, centrifuged at 8,000×g for 1 minute, andthe filtrate was discarded. This procedure was repeated twice. Finally,an upper filter part was transferred to a fresh sample tube, supplied 30μL of RNase-free sterilized water attached to the kit, and centrifugedat 8,000×g for 1 minute, and total RNA was dissolved out into thefiltrate. This dissolution procedure was repeated twice. Theconcentration of the resulting total RNA solution was obtained from theabsorbance at 260 nm to be 322 μg/ml.

Then, a cDNA was synthesized using ThermoScript RT-PCR System(Invitrogen) while employing total RNA as a template. A solution inwhich 2.7 μl, of total RNA and 6.3 μL of sterilized distilled water weremixed into 1.0 μL of 50 mM Oligo(dt)₂₀ attached to the kit and 2.0 μL of10 mM dNTP Mix was treated at 65° C. for 5 minute, and then rapidlycooled on ice. To this solution were added 4 μL of 5×cDNA SynthesisBuffer attached to the kit, 1 μL of 0.1M DTT, 1 μL of RNase OUT, 1 μL ofThermoScript RT and 1 μL of sterilized distilled water, to react them at50° C. for 60 minutes and, thereafter, the reaction was stopped byheating treatment at 85° C. for 5 minutes. Further, a RNA of a templatewas degraded by adding 1 μL of RNaseH attached to the kit to thisreaction solution and maintained a temperature at 37° C. for 20 minutes,to obtain a cDNA.

A DNA having a nucleotide sequence encoding an amino acid sequence ofBotryotinia fuckiliana BcOS-1 (hereinafter, referred to as BcOS-1 DNA insome cases) was amplified by PCR using this cDNA as a template. Using anoligonucleotide comprising the nucleotide sequence represented by SEQ IDNO: 3 and an oligonucleotide consisting of the nucleotide sequencerepresented by SEQ ID NO: 4 as a primer, a PCR was performed to amplifya DNA having the nucleotide sequence represented by SEQ ID NO: 2. ThePCR was performed using KOD-Plus- (TOYOBO) under the amplifyingconditions that a temperature was maintained at 94° C. for 2 minutesand, thereafter, 35 cycles of incubation were repeated, each cyclecomprising maintaining a temperature at 94° C. for 15 seconds, then, at55° C. for 30 seconds, then, at 68° C. for 6 minutes. The PCR reactionsolution (50 μL) was prepared by adding 2 μL of the aforementioned cDNA,5 μL of 10× Buffer, 5 μL of 2 mM dNTPs, 2 μL of 25 mM MgSO₄, each 1 μLof 10 μM oligonucleotide primers, 33 μL of sterilized distilled waterand 1 μL of KOD-Plus-. After the reaction, a part of the reactionsolution was separated by 0.8% agarose gel electrophoresis, and stainedwith ethidium bromide. It was confirmed that about 4 kb of a DNA (BcOS-1DNA) was amplified.

Example 2 Construction of Expression Plasmid of Botryotinia fuckelianaBcOS-1 Gene and Preparation of Transformed Budding Yeast

BcOS-1 DNA was cloned into a shuttle vector p415ADH (ATCC87312)replicable in yeast and Escherichia coli. About 4 kb of theaforementioned DNA (BcOS-1 DNA) was purified from the PCR reactionsolution prepared in Example 1 using QIAquick PCR Purification Kit(QIAGEN) according to the attached manual. About 4 kb of the purifiedDNA (BcOS-1 DNA) was digested with restriction enzymes SpeI and PstIand, on the other hand, the shuttle vector p415ADH was also digestedwith restriction enzymes SpeI and PstI and, thereafter, each of whichwas separated by 0.8% agarose gel electrophoresis, and a part of the gelcontaining a desired DNA was excised. The BcOS-1 DNA digested with SpeIand PstI and the shuttle vector digested with SpeI and PstI wererecovered from the gel using QIAquick Gel Extraction Kit (QIAGEN)according to the attached manual. The aforementioned BcOS-1 DNA wasinserted between SpeI site and PstI site in the multicloning site of theshuttle vector using Ligation Kit Ver. 2 (TaKaRa) according to theattached manual, to construct an expression plasmid pADHBcOS1. Anucleotide sequence of the resulting expression plasmid was analyzedwith a DNA sequencer (Model 3100, Applied Biosystems) after a sequencingreaction using BigDye terminator v3.0 Cycle Sequencing Ready ReactionKit (Applied Biosystems) according to the attached manual. Thesequencing reaction was performed using an oligonucleotide consisting ofthe nucleotide sequence represented by any of SEQ ID NOs: 5 to 12 as aprimer under the amplifying conditions that 30 cycles were repeated,each cycle comprising maintaining a temperature at 96° C. for 10seconds, then, at 50° C. for 5 seconds, then, at 60° C. for 4 minutes.As a result, the nucleotide sequence represented by SEQ ID NO: 2 wasobtained, and it was confirmed that the expression plasmid pADHBcOS1harbored a DNA having a nucleotide sequence encoding an amino acidsequence of BcOS-1.

The prepared expression plasmid pADHBcOS1 was introduced into each ofbudding yeast (Saccharomyces cerevisiae) AH22 strain (IFO10144) andTM182 strain (Maeda T. et al. (1994) Nature vol. 369, pp 242-245)according to the method described in Geitz R D & Woods R A (1994)Molecular Genetics of Yeast: Practical Approaches ed. Johnson J A,Oxford University Press pp 124-134. By utilizing disappearance ofleucine auxotrophy in the resulting transformed budding yeast, thetransformed budding yeast AH 22 strain (AH22-BcOS1) was selected on aGlu-Leu agar medium, and the transformed budding yeast TM182 strain(TM182-BcOS1) was selected on a Gal-Ura-Leu agar medium. It wasconfirmed that the resulting TM182-BcOS1 grows even when transplanted toa Glu-Ura-Leu medium.

Example 3 Antifungal Compound Sensitivity Test of Transformed BuddingYeast TM182-BcOS1

The transformed budding yeast AH22-BcOS1 prepared in Example 2 wascultured while shaking at 30° C. for 18 hours in a Glu-Leu medium. As acontrol, the AH22 strain was similarly cultured while shaking at 30° C.for 18 hours in a Glu medium. The absorbance at 600 nm of each of thegrown transformed budding yeasts in a cell suspension was measured, anda cell suspension diluted with sterilized distilled water to theabsorbance of 0.1 was prepared. Further, a cell suspension in which theaforementioned cell suspension of the transformed budding yeastAH22-BcOS1 was diluted 200-fold with a Glu-Leu medium, and a cellsuspension in which the aforementioned cell suspension of the AH22strain was diluted 200-fold with a Glu medium were prepared. A solutionin which each of Compounds (1) to (3) was dissolved in dimethylsulfoxide(DMSO) to the concentration of 60 ppm, a solution in which each ofCompounds (4) and (5) was dissolved in dimethylsulfoxide (DMSO) to theconcentration of 2000 ppm, and a solution in which each of Compounds (6)and (7) was dissolved in dimethylsulfoxide (DMSO) to the concentrationof 20 ppm were prepared, and two microplates were prepared in which each2.0 μL per well of the Compound solution and DMSO as a control weredispensed into two wells. In one microplate among them, each 200 μL ofcell suspensions of the transformed budding yeast AH22-BcOS1 which hadbeen prepared by dilution as described above was dispensed, and culturedby allowing to stand at 30° C. for 48 hours. In another microplate, each200 μL of the cell suspensions of the control yeast AH22 strain whichhad been prepared by dilution as described above was dispensed, andcultured by allowing to stand at 30° C. for 48 hours. After culturing,the absorbance at 600 nm of each well was measured with a microplatereader.

Similarly, the transformed budding yeast TM182-BcOS1 prepared in Example2 was cultured at 30° C. for 18 hours in a Glu-Ura-Leu medium. Theabsorbance at 600 nm of the grown transformed budding yeast in a cellsuspension was measured, and a cell suspension diluted with sterilizeddistilled water to the absorbance of 0.1 was prepared. Further, a cellsuspension in which the aforementioned cell suspension of thetransformed budding yeast TM182-BcOS1 was diluted 200-fold with aGlu-Ura-Leu medium and, as a control, a cell suspension in which theaforementioned cell suspension was diluted 200-fold with a Gal-Ura-Leumedium were prepared. A suspension in which each of Compounds (1) to (3)was dissolved in dimethylsulfoxide (DMSO) to the concentration of 60ppm, a solution in which each of Compounds (4) and (5) was dissolved indimethylsulfoxide (DMSO) to the concentration of 2000 ppm, and asolution in which each of Compounds (6) and (7) was dissolved indimethylsulfoxide (DMSO) to the concentration of 20 ppm were prepared,and two microplates were prepared in which each 2.0 μL per well of theCompound solution and DMSO as a control were dispensed into two wells.In one microplate among them, each 200 μL of cell suspensions of thetransformed budding yeast TM182-BcOS1 which had been prepared bydilution with a Glu-Ura-Leu medium as described above was dispensed, andcultured by allowing to stand at 30° C. for 67 hours. In anothermicroplate, as described above, as a control, each 200 μL of the cellsuspensions of the transformed budding yeast TM182-BcOS1 which had beenprepared by dilution with a Gal-Ura-Leu medium was dispensed, andcultured by allowing to stand at 30° C. for 67 hours. After culturing,the absorbance at 600 nm of each well was measured with a microplatereader.

Degree of growths of both of the transformed budding yeasts culturedunder the presence of each of Compound (1) to (7) and budding yeast as acontrol therefor are shown in Table 1. Degree of growths of both of thetransformed budding yeasts and budding yeasts as a control therefor areexpressed by a relative value in percentage, letting the absorbance at600 nm in a well having the concentration of the aforementioned Compoundof 0 ppm to be 100. It was confirmed that an inhibiting degree of growthof TM182-BcOS1 by each test substance was grater than an inhibitingdegree of growth of AH22-BcOS1 by each test substance, and theTM182-BcOS1 was a transformed cell with the enhanced sensitivity to anantifungal compound as compared with AH22-BcOS1.

TABLE 1 Degree of growth of budding yeast (%) AH22- AH22 BcOS1TM182-BcOS1 Test substance Glu Glu-Leu Gal-Ura- Gal-Ura- (finalconcentration) medium medium Leu medium Leu medium Compound (1) (0.6ppm) 99 90 99  9 Compound (2) (0.6 ppm) 99 92 98 11 Compound (3) (0.6ppm) 98 93 98 10 Compound (4) (20 ppm) 96 45 102  10 Compound (5) (20ppm) 97 79 103  48 Compound (6) (0.2 ppm) 99 81 99  8 Compound (7) (0.2ppm) 101  94 99 11

Example 4 Isolation of Botryotinia fuckeliana Mutant BcOS-1 GeneExhibiting Resistance to Dicarboxyimide Antifungal Compound

A DNA having a nucleotide sequence encoding an amino acid sequence ofBotryotinia fuckeliana mutant BcOS-1 (Oshima, M. et al. (2002)Phytopathology 92, pp 75-80) exhibiting resistance to a dicarboxyimideantifungal compound (hereinafter, referred to as mutant BoOS1 DNA insome cases) was prepared by PCR using the cDNA prepared in Example 1 asa template. A first time PCR was performed using, as a primer, anoligonucleotide consisting of the nucleotide sequence represented by SEQID NO: 15 and an oligonucleotide consisting of the nucleotide sequencerepresented by SEQ ID NO: 4, and a DNA having a nucleotide sequencerepresented by base numbers 1081 to 3948 of the nucleotide sequencerepresented by SEQ ID NO: 14 was amplified. The PCR was performed usingKOD-Plus- (TOYOBO) under the amplifying conditions that a temperaturewas maintained at 94° C. for 2 minutes and, thereafter, 35 cycles wererepeated, each cycle comprising maintaining a temperature at 94° C. for15 seconds, then, at 55° C. for 30 seconds, then, at 68° C. for 6minutes. The PCR reaction solution (50 μl) was prepared by adding 2 μLof the aforementioned cDNA, 5 μL of 10× Buffer, 5 μL of 2 mM dNTPs, 2 μLof 25 mM MgSO₄, each 1 μL of 10 μM oligonucleotide primers, 33 μL ofsterilized distilled water and 1 μL of KOD-Plus-. After the reaction, asecond PCR was performed using an oligonucleotide consisting of thenucleotide sequence represented by SEQ ID NO: 3 and 1 μL of the firsttime PCR reaction solution while using the cDNA prepared in Example 1 asa template. The reaction conditions were the same as those of the firsttime PCR and after the reaction, a part of the reaction solution wasseparated by 0.8% agarose gel electrophoresis, and stained with ethidiumbromide. It was confirmed that about 4 kb of the DNA (mutant BcOS-1 DNA)was amplified.

Example 5 Construction of Expression Plasmid of Botryotinia fuckelianaBcOS-1 Mutant Gene Exhibiting Resistance to Dicarboxyimde AntifungalCompound and Preparation of Transformed Budding Yeast

First, the mutant BcOS-1 DNA was cloned into a vector pBluescript IISK(+) (TOYOBO). About 4 kb of the DNA (mutant BcOS-DNA) was purifiedfrom the second time PCR reaction solution prepared in Example 4 usingQIAquick PCR Purification Kit (QIAGEN) according to the attached manual.About 4 kb of the purified DNA (mutant BcOS-1 DNA) was digested withrestriction enzymes SpeI and PstI and, on the other hand, the vectorpBluescript II SK(+) was also digested with restriction enzymes SpeI andPstI, each of which was separated by 0.8% agarose gel electrophoresis,and a part of the gel containing a desired DNA was excised. The mutantBcOS-1 DNA digested with SpeI and PstI and the vector pBluescript IISK(+) digested with SpeI and PstI were recovered from the gel usingQIAquick Gel Extraction Kit (QIAGEN) according to the attached manual.The aforementioned mutant BcOS-1 DNA was inserted between SpeI site andPstI site in the multicloning site of the vector pBluescript II SK(+)using Ligation Kit Ver. 2 (TaKaRa) according to the attached manual, toconstruct a plasmid pBcOS1-I 365S. A nucleotide sequence of theresulting plasmid was analyzed with a DNA sequencer (Model 3100, AppliedBiosystems) after a sequencing reaction using BigDye terminator v3.0Cycle Sequence FS Ready Reaction Kit (Applied Biosystems) according tothe attached manual. The sequencing reaction was performed by using anoligonucleotide consisting of the nucleotide sequences represented byany of SEQ ID NOs: 7 to 12 as a primer under the amplifying conditionsthat 30 cycles were repeated, each cycle comprising maintaining atemperature at 96° C. for 10 seconds, then, at 50° C. for 5 seconds,then, at 60° C. for 4 minutes. As a result, the nucleotide sequencerepresented by SEQ ID NO: 14 was obtained and it was confirmed that theplasmid pBcOS1-I 365S harbored the mutant BcOS-1 DNA.

The mutant BcOS-1 DNA contained in the thus prepared plasmidpBcOS1-I365S was cloned into a shuttle vector p415ADH replicable inyeast and Escherichia coli, to construct an expression plasmid. Theplasmid pBcOS1-I365S was digested with restriction enzymes SpeI and PstIand, on the other hand, the shuttle vector p415ADH was also digestedwith restriction enzymes SpeI and PstI. These were separated by 0.8%agarose gel electrophoresis, respectively, each of gel parts containingthe mutant BcOS-1 DNA digested with SpeI and PstI and the shuttle vectorp415ADH digested with SpeI and PstI was excised, and the mutant BcOS-1DNA and the shuttle vector were recovered from the gel using QIAquickGelExtraction Kit (QIAGEN) according to the attached manual. The mutantBcOS-1 DNA was inserted between SpeI site and PstI site in themulticloning site of the shuttle vector using Ligation Kit Ver. 2(TaKaRa) according to the attached manual, to construct an expressionplasmid pADHBcOS1-I365S. A nucleotide sequence of the resultingexpression plasmid was analyzed with a DNA sequencer (Model 3100,Applied Biosystems) after a sequencing reaction using BigDye terminatorv3.0 Cycle Sequence FS Ready Reaction Kit (Applied Biosystems) accordingto the attached manual. The sequencing reaction was performed by usingan oligonucleotide consisting of the nucleotide sequence represented byany of SEQ ID NOs: 5 to 12 as a primer under the amplifying conditionsthat 30 cycles of incubation were repeated, each cycle comprisingmaintaining a temperature at 96° C. for 10 seconds, then, at 50° C. for5 seconds, then, at 60° C. for 4 minutes. As a result, the nucleotidesequence represented by SEQ ID NO: 14 was obtained, and it was confirmedthat the expression plasmid pADHBcOS1-I365S harbored a DNA having anucleotide sequence encoding an amino acid sequence of the mutantBcOS-1.

The prepared expression plasmid pADHBcOS1-I 365S was introduced into thebudding yeast TM182 strain according to the method described in Example2. By utilizing disappearance of leucine auxotrophy in the resultingtransformed budding yeast, the transformed budding yeast TM182 strain(TM182-BcOS1-I365s) was selected on a Gal-Ura-Leu agarose medium. It wasconfirmed that the resulting TM182-BcOS1-I365S grows even whentransplanted to a Glu-Ura-Leu medium.

Example 6 Antifungal Compound Sensitivity Test of Transformed BuddingYeast TM182-BcOS1-I-365S

The transformed budding yeast TM182-BcOS1-I365S prepared in Example 5was cultured at 30° C. for 18 hours in a Glu-Ura-Leu medium. Theabsorbance at 600 nm of a cell suspension of the grown transformedbudding yeast was measured, and a cell suspension diluted withsterilized distilled water to the absorbance of 0.1 was prepared.Further, a cell suspension in which the aforementioned cell suspensionof the transformed budding yeast TM182-BcOS1-I 365S was diluted 200-foldwith a Glu-Ura-Leu medium and, as a control, a cell suspension in whichthe cell suspension was diluted 200-fold with a Gal-Ura-Leu medium wereprepared. A solution in which each of Compound (1) to (3) was dissolvedin dimethylsulfoxide (DMSO) to the concentration of 60 ppm, a solutionin which each of Compounds (4) and (5) was dissolved indimethylsulfoxide (DMSO) to the concentration of 2000 ppm, and asolution in which each of Compounds (6) and (7) was dissolved indimethylsulfoxide (DMSO) to the concentration of 20 ppm were prepared,and two microplates were prepared in which each 2.0 μL per well of theCompound solution and DMSO as a control were dispensed into two wells.In one microplate among them, each 200 μL of cell suspensions of thetransformed budding yeast TM182-BcOS1-I365S which had been prepared bydilution with a Glu-Ura-Leu medium as described above was dispensed, andcultured by allowing to stand at 30° C. for 67 hours. In anothermicroplate, as a control, each 200 μL of cell suspensions of thetransformed budding yeast TM182-BcOS1-I 365S which had been prepared bydilution with a Gal-Ura-Leu medium was dispensed, and cultured byallowing to stand at 30° C. for 67 hours. After culturing, theabsorbance at 600 nm of each well was measured with a microplate reader.

Degrees of growths of both of the transformed budding yeasts culturedunder the presence of Compounds (1) to (7) and budding yeast as acontrol therefor are shown in Table 2. Degrees of growths of both of thetransformed budding yeasts and budding yeast as a control are expressedby a relative value in percentage, letting the absorbance at 600 nm atthe concentration of the Compound of 0 ppm to be 100. It was confirmedthat an inhibiting degree of growth of the transformed budding yeastTM182-BcOS1-I 365S by each test substance was grater than an inhibitingdegree of growth of the transformed budding yeast AH22-BcOS1-I 365S byeach test substance, and the transformed budding yeast TM182-BcOS1-I365S was a transformed cell with the enhanced sensitivity to anantifungal compound as compared with the transformed budding yeastAH22-BcOS1-I365S.

TABLE 2 Degree of growth of budding yeast (%) AH22- BcOS1- AH22 I365STM182-BcOS1-I365S Test substance Glu Glu-Leu Gal-Ura- Glu-Ura- (finalconcentration) medium medium Leu medium Leu medium Compound (1) (6 ppm)88 68 99  9 Compound (2) (6 ppm) 91 81 88 11 Compound (3) (6 ppm) 87 7592  9 Compound (4) (20 ppm) 96 83 101  41 Compound (5) (20 ppm) 80 64 7613 Compound (6) (0.2 ppm) 92 67 93  7 Compound (7) (0.2 ppm) 91 79 90 22

Example 7 Isolation of Magnaporthe grisea HIK1 Gene

Total RNA was prepared from Magnaporthe grisea. 100 mg of a hypha ofMagnaporthe grisea P-37 strain which had been grown on a potato dextroseagar medium (PDA medium manufactured by NISSUI Pharmaceutical Co., Ltd.)was scratched off, and this was ground using a mortar and a pestle inliquid nitrogen. A RNA was prepared from frozen ground powder usingRNeasy Plant Mini Kit (QIAGEN). A frozen ground powder together withliquid nitrogen was transformed to a 50 ml sample tube and, after liquidnitrogen was all volatilized off, a solution obtained by adding 10 μL ofmercaptoethanol was added per 1 ml of a buffer RLC attached to the kitwas added, followed by stirring. Further, after ground powder was welldispersed by a few pipettings, a temperature was maintained at 56° C.for 3 minutes. Thereafter, a solution containing ground powder wassupplied to QIAshredder spin column attached to the kit, and centrifugedat 8,000×g for 2 minutes. The filtration supernatant was transferred toa fresh sample tube, a 0.5-fold volume of 99.5% ethanol was added, andthe material was well mixed by pipetting. This mixtured solution wassupplied to RNeasy mini spin column attached to the kit, and centrifugedat 8,000×g for 1 minute. The filtrate was discarded, 700 μL of BufferRW1 attached to the kit was added, centrifuged at 8,000×g for 1 minute,and the filtrate was discarded. Further, the residue was added 500 μL ofBuffer RPE attached to the kit, and centrifuged at 8,000×g for 1 minute,and the filtrate was discarded. This procedure was repeated twice.Finally, an upper filter part was transferred to a fresh sample tube,supplied 30 μL of RNase-free sterilized water, and centrifuged at8,000×g for 1 minute, and total RNA was dissolved into the filtrate.This dissolution procedure was repeated twice.

Then, a cDNA was synthesized using ThermoScript RT-PCR System(Invitrogen) while using total RNA as a template. A solution in which9.0 μL of total RNA was mixed into 1.0 μL of 50 mM Oligo(dt)₂₀ attachedto the kit and 2.0 μL of 10 mM dNTP Mix was treated at 65° C. for 5minutes, and rapidly cooled on ice. To this solution were added 4 μL of5×cDNA Synthesis Buffer attached to the kit, 1 μL of 0.1M DTT, 1 μL ofRNase OUT, 1 μL of ThermoScript RT and 1 μl of sterilized distilledwater, to react them at 50° C. for 60 minutes and, thereafter, thereaction was stopped by heating treatment at 85° C. for 5 minutes.Further, 1 μL of RNaseH attached to the kit was added to this reactionsolution, the materials were reacted at 37° C. for 20 minutes, and a RNAas a template was degraded to obtain a cDNA.

A DNA having a nucleotide sequence encoding an amino acid sequence ofMagnaporthe grisea HIK1 (hereinafter, referred to as HIK1 DNA in somecases) was amplified by PCR using this cDNA as a template. A PCR wasperformed using an oligonucleotide consisting of the nucleotide sequencerepresented by SEQ ID NO: 18 and an oligonucleotide consisting of thenucleotide sequence represented by SEQ ID NO: 19, to amplify a DNAhaving the nucleotide sequence represented by SEQ ID NO: 17. The PCR wasperformed using KOD-Plus- (TOYOBO) under the amplifying conditions thata temperature was maintained at 94° C. for 2 minutes and, thereafter, 35cycles of incubation were repeated, each cycle comprising maintaining atemperature at 94° C. for 15 seconds, then, at 55° C. for 30 seconds,then, at 68° C. for 6 minutes. The PCR reaction solution (50 μL) wasprepared by adding 2 μL of the aforementioned cDNA, 5 μL of 10× Buffer,5 μL of 2 mM dDNPs, 2 μL of 25 mM MgSO₄, each 1 μL of 10 μMoligonucleotide primers, 33 μL of sterilized distilled water and 1 μL ofKOD-Plus-. After the reaction, a part of the reaction solution wasseparated with 1.0% agarose gel electrophoresis, and stained withethidium bromide. It was confirmed that about 4 kb of the DNA (HIK1 DNA)was amplified.

Example 8 Construction of an Expression Plasmid of Magnaporthe griseaHIK1 Gene and Preparation of Transformed Budding Yeast

The HIK1 DNA was cloned into a cloning vector pBluesripit SK II(+).About 4 kb of the aforementioned. DNA (HIK1 DNA) was purified from thePCR reaction solution prepared in Example 7 using QIAquick PCRPurification Kit (QIAGEN) according to the attached manual. About 4 kbof the purified DNA (HIK1 DNA) was digested with restriction enzymes ofSpeI and HindIII and, on the other hand, after the cloning vectorpBluescript SK II (t) (manufactured by Stratagene) was also digestedwith restriction enzymes SpeI and HindIII, each of which was separatedwith 1.0% agarose gel electrophoresis, and a part of the gel containinga desired DNA was excised. The NIK1 DNA digested with SpeI and HindIIIand the cloning vector digested with SpeI and HindIII were recoveredform the gel using QIAquick Gel Extraction Kit (QIAGEN) according to theattached manual. The HIK1 DNA was inserted between SpeI site and HindIIIsite in the multicloning site of the cloning vector using Ligation KitVer. 2 (TaKaRa) according to the attached manual, to construct a plasmidpBlueHIK1. A nucleotide sequence of the resulting plasmid was analyzedwith a DNA sequencer (Model 3100, Applied Biosystems) after a sequencingreaction using BigDye terminator v3.0 Cycle Sequence FS Ready ReactionKit (Applied Biosystems) according to the attached manual. Thesequencing reaction was performed using an oligonucleotide consisting ofthe nucleotide sequence represented by any of SEQ ID NOs:20 to 29 as aprimer under the amplifying conditions that 35 cycles of incubation wererepeated, each cycle comprising maintaining a temperature at 96° C. for10 seconds, then, at 50° C. for 5 seconds, then, at 60° C. for 2minutes. As a result, the nucleotide sequence represented by SEQ ID NO:17 was obtained, and it was confirmed that the plasmid pBlueHIK1harbored a DNA having a nucleotide sequence encoding an amino acidsequence of HIK1.

Then, the HIK1 DNA was inserted into a shuttle vector p415ADH(ATCC87312) replicable in yeast and Escherichia coli. The plasmidpBlueHIK1 prepared as described above was digested with restrictionenzymes SpeI and HindIII and, on the other hand, after the shuttlevector p415ADH (ATCC87312) was also digested with restriction enzymesSpeI and HindIII, each of which was separated with 1.0% agarose gelelectrophoresis, and a part of the gel containing a desired DNA wasexcised. The HIK1 DNA digested with SpeI and HindIII and the shuttlevector digested with SpeI and HindIII were recovered from the gel usingQIAquick Gel Extraction Kit (QIAGEN) according to the attached manual.The HIK1 DNA was inserted between SpeI site and HindIII site in themulticloning site of the shuttle vector using Ligation Kit Ver. 2(TaKaRa) according to the attached manual, to construct an expressionplasmid pADHHIK1.

The prepared expressed plasmid pADHHIK1 was introduced into buddingyeast (Saccharomyces cerevisiae) AH22 strain (IFO10144) and TM182 strain(Maeda T. et al. (1994) Nature vol. 369, pp 242-245) according to themethod described in Geitz R D & Woods R A (1994) Molecular Genetics ofYeast: Practical Approaches ed. Johnson J A, Oxford University Press pp124-134. By utilizing disappearance of leucine auxotrophy in theresulting transformed budding yeast, the transformed budding yeast AH22strain (AH21-HIK1) was selected on a Glu-Leu agar medium, and thetransformed budding yeast TM182 strain (TM182-HIK1) was selected on aGlu-Ura-Leu agar medium. It was confirmed that the resulting TM182-HIK1grows even transferred to a Glu-Ura-Leu medium.

Example 9 Antifungal Compound Sensitivity Test of Transformed BuddingYeast TM182-HIK1

The transformed budding yeast AH22-HIK1 prepared in Example 8 wascultured while shaking at 30° C. for 24 hours in a Glu-Leu medium. As acontrol, the AH22 strain was similarly cultured while shaking at 30° C.for 24 hours in a Glu medium. The absorbance at 600 nm of a cellsuspension of each of the grown transformed budding yeasts was measured,and a cell suspension diluted with each medium to the absorbance of 0.1was prepared. Further, a cell suspension in which the aforementionedcell suspension of the transformed budding yeast AH22-HIK1 was diluted50-fold with a Glu-Leu medium, and a cell suspension in which theaforementioned cell suspension of AH22 strain was diluted 50-fold with aGlu medium were prepared. A suspension in which each of compounds (1) to(3) was dissolved in dimethylsulfoxide (DMSO) to the concentration of200 ppm, a solution in which each of Compounds (4) to (5) was dissolvedin dimethylsulfoxide (DMSO) to the concentration of 600 ppm, and asolution in which each of Compounds (6) and (7) was dissolved indimethylsulfoxide (DMSO) to the concentration of 20 ppm were prepared,and two microplates were prepared in which each 1.0 μL per well of theCompound solution and DMSO as a control were dispensed into two wells.In one microplate among them, each 100 of a cell suspension of thetransformed budding yeast AH22-hiki which had been prepared by dilutionas described above was dispensed, and cultured by allowing to stand at30° C. for 23 hours. In another microplate, each 100 μL of the cellsuspensions of control yeast AH22 strain which had been prepared bydilution as described above was dispensed, and cultured by allowing tostand at 30° C. for 27 hours. After culturing, the absorbance at 600 nmof each well was measured with a microplate reader.

Similarly, the transformed budding yeast TM182-HIK1 prepared in Example8 was cultured at 30° C. for 24 hours in a Glu-Ura-Leu medium. Theabsorbance at 600 nm of a cell suspension of the grown transformedbudding yeast was measured, and a cell suspension diluted with eachmedium to the absorbance of 0.1 was prepared. Further, a cell suspensionin which the aforementioned cell suspension of the transformed buddingyeast TM182-HIK1 was diluted 50-fold with a Glu-Ura-Leu medium and, as acontrol, a cell suspension in which the suspension was diluted 50-foldwith a Glu-Ura-Leu medium were prepared. A suspension in which each ofCompounds (1) to (3) was dissolved in dimethylsulfoxide (DMSO) to theconcentration of 200 ppm, a solution in which each of Compounds (4) and(5) was dissolved in dimethylsulfoxide (DMSO) to the concentration of600 ppm, and a solution in which each of Compounds (6) and (7) wasdissolved in dimethylsulfoxide (DMSO) to the concentration of 20 ppmwere prepared, and two microplates were prepared in which each 1.0 μLper well of the Compound DMSO solution and DMSO as a control weredispensed into two wells. In one microplate among them, each 100 μL ofcell suspensions of the transformed budding yeast TM182-HIK1 which hadbeen prepared by dilution with a Glu-Ura-Leu medium as described abovewas dispensed, and cultured by allowing to stand at 30° C. for 27 hours.In another microplate, as described above, as a control, each 100 μL ofcell suspensions of the transformed budding yeast TM182-HIK1 which hadbeen prepared by dilution with a Gal-Ura-Leu medium was dispensed, andcultured by allowing to stand at 30° C. for 27 hours. After culturing,the absorbance at 600 nm of each well was measured with a microplatereader.

Degree of growths of both of the transformed budding yeasts cultured inthe presence of Compounds (1) to (7) and budding yeast as a controltherefor are shown in Table 3. Degrees of growths of both of thetransformed budding yeasts and budding yeast as a control thereofor areshown by a relative value in percentage, letting the absorbance of 600nm at the concentration of the Compound of 0 ppm to be 100. It wasconfirmed that an inhibiting degree of growth of TM182-HIK1 by each testsubstance was greater than an inhibiting degree of growth of AH22-HIK1by each test substance, and the TM182-HIK1 was a transformed cell withthe enhanced sensitivity to an antifungal compound as compared withAH22-HIK1.

TABLE 3 Degree of growth of budding yeast (%) AH22- AH22 HIK1 TM182-HIK1Test substance Glu Glu-Leu Gal-Ura- Glu-Ura- (final concentration)medium medium Leu medium Leu medium Compound (1) (2.0 ppm) 85 89 100 62Compound (2) (2.0 ppm) 96 84  94 79 Compound (3) (2.0 ppm) 99 104  10030 Compound (4) (6.0 ppm) 97 92  97 63 Compound (5) (6.0 ppm) 93 99 10622 Compound (6) (0.2 ppm) 101  98 104 11 Compound (7) (0.2 ppm) 89 102  87  9

Example 10 Amplification of Osmosensitivie Histidine Kinase GeneFragment from Other Filamentas Fungus

(1) Preparation of Total RNA of Fusarium oxysporum

Total RNA was prepared from Fusarium oxysporum. 100 mg of a hypha ofFusarium oxyporum RJN1 strain grown on a potato dextrose agarose medium(PDA medium, manufactured by NISSUI Pharmaceutical Co., Ltd.) wascollected, and this was ground using a mortar and a pestle in liquidnitrogen. Total RNA was prepared from frozen ground powder using RNeasyPlant Mini Kit (QIAGEN) according to the method described in Example 1.

(2) Preparation of Total RNA of Mycospharella tritici

Total RNA was prepared from Mycospharella tritici. Spore ofMycospharella tritici St-8 strain grown on a potato dextrose agarosemedium (PDA medium, manufactured by NISSUI Pharmaceutical Co., Ltd.) wasadded to 100 ml of PD broth (DIFCO), and this was cultured at 20° C. and150 rpm for 4 days using a 500 ml volume Erlenmeyer flask. 8 ml of theculture solution was centrifuged to remove the supernatant, and 300 mgof a wet weight of cells were transferred to a mortar and ground inliquid nitrogen using a pestle. Total RNA was prepared from frozenground powder according to the method described in Example 1.

(3) Preparation of Total RNA of Thanatephorus cucumeris

Total RNA was prepared from Thanatephorus cucumeris. Hypha ofThanatephorus cucumeris Rs-18 strain grown on a potato dextrose agarmedium (PDA medium, manufactured by NISSUI Pharmaceutical Co., Ltd.) wasadded to 100 ml of PD broth (DIFCO), and cultured by allowing to standat 25° C. for 4 days using a 500 ml volume Erlenmeyer flask. 8 ml of theculture solution was centrifuged to remove the supernatant, 300 mg of awet weight of hypha were transferred to a mortar, and ground in liquidnitrogen using a pestle. Total RNA was prepared from frozen groundpowder using Rneasy Plant Mini Kit (QIAGEN) according to the methoddescribed in Example 1.

(4) Preparation of Total RNA of Phytophthora infestans

Total RNA was prepared from Phytophthora infestans. Hypha ofPhytophthora infestans Pi-5 strain grown on a rye agar medium (rye 60 g,sucrose 15 g, agar 20 g/1 L) was added to 20 ml of a rye medium (rye 60g, sucrose 15 g/1 L), and cultured at 20° C. and 150 rpm for 5 daysusing a 300 ml of volume Erlenmeyer flask. 20 ml of the culture solutionwas centrifuged to remove the supernatant, a wet weight of 200 mg ofcells were transferred to a mortar, and ground using a pestle in liquidnitrogen. Total RNA was prepared from frozen ground powder using RNeasyPlant Mini Kit (QIAGEN) according to the method described in Example 1.

(5) Amplification of Osmosensing Histidine Kinase Gene Fragment by PCR

Using the total RNA of Magnaporthe grisea prepared in Example 7, thetotal RNA of Fusarium oxysporum prepared in Example 10 (1), the totalRNA of Mycospharella tritici prepared in Example 10 (2), the total RNAof Thanatephorus cucumeris prepared in Example 10 (3), or the total RNAof Phytophthora infestans prepared in Example 10 (4), amplification of aDNA having a nucleotide sequence encoding a part of an amino acidsequence of osmosensing histidine kinase was performed.

First, a cDNA was synthesized using ThermoScript RT-PCR System(Invitrogen) and using each of total RNAs as a template. A solution inwhich 4.0 μL of each of total RNAs and 5.0 μL of sterilized distilledwater were mixed into 1.0 μL of 50 mM Oligo(dT)₂₀ attached to the kitand 2.0 μL of 10 mM dNTP Mix was prepared, and a cDNA was synthesizedaccording to the method described in Example 1.

A PCR was performed using each cDNA as a template. As primers, a primerpair shown in Table 4 was used. A size of a DNA which is predicted to beamplified by PCR using each primer pair based on the nucleotide sequencerepresented by SEQ ID NO: 2 is shown in Table 4.

TABLE 4 DNA to be Primer Pair Primer Primer amplified 1 SEQ ID NO: 30SEQ ID NO: 35  368 bp 2 SEQ ID NO: 30 SEQ ID NO: 36  374 bp 3 SEQ ID NO:30 SEQ ID NO: 37  383 bp 4 SEQ ID NO: 31 SEQ ID NO: 35  359 bp 5 SEQ IDNO: 31 SEQ ID NO: 36  365 bp 6 SEQ ID NO: 31 SEQ ID NO: 37  374 bp 7 SEQID NO: 32 SEQ ID NO: 38 3019 bp 8 SEQ ID NO: 32 SEQ ID NO: 40 3052 bp 9SEQ ID NO: 33 SEQ ID NO: 38 2927 bp 10 SEQ ID NO: 33 SEQ ID NO: 40 2960bp 11 SEQ ID NO: 34 SEQ ID NO: 38 2867 bp 12 SEQ ID NO: 34 SEQ ID NO: 402900 bp 13 SEQ ID NO: 30 SEQ ID NO: 39 1424 bp 14 SEQ ID NO: 30 SEQ IDNO: 40 1442 bp 15 SEQ ID NO: 31 SEQ ID NO: 39 1415 bp 16 SEQ ID NO: 31SEQ ID NO: 40 1433 bp

A PCR was performed using KOD-Plus- (TOYOBO) under the amplifyingconditions that a temperature was maintained at 94° C. for 2 minutesand, thereafter, 35 cycles of incubation were repeated, each cyclecomprising maintaining a temperature at 94° C. for 15 seconds, then, at55° C. for 30 seconds further, at 68° C. for 1 minutes. When primerpairs 1 to 6 were used, the incubation at 68° C. in the cycle was for 1minutes. When the primer pairs 7 to 12 were used, the incubation at 68°C. in the cycle was for 5 minutes. When the primer pairs 13 to 16 wereused, the incubation at 68° C. in the cycle was for 3 minutes. The PCRreaction solution (25 μL) was prepared by adding 0.5 μL of the cDNA, 2.5μL of 10× buffer, 2.5 μL of 8 mM dNTPs, 1.0 μL of 25 mM MgSO₄, each 0.5μL of 10 μM oligonucleotide primers, 17 μL of sterilized distilled waterand 0.5 μL of KOD-Plus-. The PCR reaction solution after the reactionwas analyzed with 1% or 4% agarose gel electrophoresis.

When primer pairs 1, 2, 3, 4, 5 or 6 were used and a cDNA of Magnaporthegrisea was used as a template, amplification of predicted size of DNAwas observed. When primer pairs 2, 3, 7, 8, 9, 10, 11 or 12 were used,and a cDNA of Fusarium oxysporum was used as a template, amplificationof a predicted size of DNA was observed. When the primer pairs 3, 5, 6,13, 14, 15 or 16 were used, and cDNA of Mycospharella Tritici was usedas a template, amplification of predicted size of DNA was observed. Whenprimer pairs 2, 3, 5 or 6 were used, and cDNA of Thanatephorus cucumeriswas used as a template, amplification of a predicted size of a DNA wasobserved. When the primer pairs 5 or 6 were used, and cDNA ofPhytophthora infestans was used as a template, amplification ofpredicted size of DNA was observed.

Example 11 Isolation of Fusarium oxysporum FoOS-1 Gene

(1) Analysis of Fusarium oxysporum FoOS-1 Gene Fragment

The amplified DNA was purified from the reaction solution of PCR whichhad been performed by using a cDNA of Fusarium oxysporum as a templateand using a primer pair 9 in Example 10 (5), using QIAquick PCRPurification Kit (QIAGEN) according to the attached instruction.

Adenine was added to the 3′-terminal of the purified DNA using Ex Taq(TaKaRa) (hereinafter, referred to as 3′A addition). The reactionsolution (20 μL) for 3′A addition was prepared by adding 15.3 μL of asolution of the aforementioned purified DNA, 2.0 μL of 10× buffer, 2.5μL of 10 mM dNPTs and 0.2 μL of Ex Taq, and this was maintained at 72°C. for 30 minutes.

Thus the 3′A-added DNA and the pCR2.1-TOPO cloning vector (Invitrogen)were ligated according to the instruction attached to the cloningvector, after that, which was introduced into Escherichia coli JM109(TaKaRa). A plasmid DNA was purified from the resulted Escherichia colitransformant using QIAprep Spin Miniprep Kit (QIAGEN). A nucleotidesequence of the plasmid DNA was analyzed with a DNA sequencer (Model3100, Applied Biosystems) after a sequencing reaction employing theresulting plasmid DNA as a template, and using an oligonucleotideconsisting of the nucleotide sequence represented by any of SEQ ID NOs:28, 29, and 45 to 48 as a primer, and using BigDye Terminator v3.0 CycleSequencing Ready Reaction Kit (Applied Biosystems Japan) according tothe instruction attached to the kit. The sequencing reaction wasperformed under the amplifying conditions that 35 cycles of incubationwere repeated, each cycle comprising maintaining a temperature at 96° C.for 10 seconds, then, at 50° C. for 5 seconds, further, at 60° C. for 2minutes. As a result, a nucleotide sequence represented by base numbers663 to 3534 of the nucleotide sequence represented by SEQ ID NO: 42 wasread.

(2) Analysis of Full Length FoOS-1 Gene of Fusarium oxysporum

A DNA having a nucleotide sequence extending toward to the 5′ upstreamregion from a nucleotide number 663 of the nucleotide sequencerepresented by SEQ ID Mo. 42 was cloned using SMART RACE cDNAAmplification Kit (CLONTECH) according to the instruction attached tothe kit. 1.0 μL of CDS-primer attached to the kit, and 1.0 μL of SMARTIIA Oligo were mixed into 3 μL (230 ng) of the total RNA prepared inExample 10 (1) to prepare a reaction solution. The reation solution wasmaintained at 70° C. for 2 minutes and maintained on ice for 2 minutes.To the reaction solution were added 2 μL of 5× First-Strand bufferattached to the kit, 1 μL of 20 mM DTT, 1 μL of 10 mM dNPT Mix and 1 μLof PowerScript Reverse Transcriptase and mixed, and the mixture wasmaintained at 42° C. for 1.5 hours. To the reaction solution aftertemperature maintenance was added 100 μL of Tricine-EDTA buffer attachedto the kit, and a temperature was maintained at 72° C. for 7 minutes toprepare 5′ RACE ready cDNA. PCR amplifying 5′ upstream region wasperformed by using this 5′ RACE ready cDNA as a template. A PCR reactionsolution was obtained by adding 5.0 μL of 10× Advantage 2 buffer, 1.0 μLof 10 mM dNTP Mix and 1.0 μL of 50× Advantage 2 Polymerase Mix attachedto the kit to 2.5 μL of 5′ RACE ready cDNA and mixing them, and adding5.0 μL of 10× Universal Primer A Mix attached to the kit as a primer,and 1.0 μL of a 10 μM solution of an oligonucleotide consisting of thenucleotide sequence represented by SEQ ID NO: 43, and adding sterilizeddistilled water to a total amount of 50 μL. This reaction solution wassubjected to repetition of 5 cycles of incubation, each cycle comprisingmaintaining a temperature at 94° C. for 5 seconds, then, at 72° C. for 2minutes, further repetition of 5 cycles of incubation, each cyclecomprising a maintaining a temperature at 94° C. for 5 seconds, then, at70° C. for 10 seconds, then, at 72° C. for 2 minutes, further repetitionof 25 cycles of incubation, each cycle comprising maintaining atemperature at 94° C. for 5 seconds, then, at 68° C. for 10 seconds,then, at 72° C. for 2 minutes, followed by maintaining a temperature at72° C. for 7 minutes. The PCR reaction solution and the pCR2.1-TOPOcloning vector (Invitrogen) were ligated according to the instructionattached to the cloning vector, after that, which was introduced intoEscherichia coli JM109 (TaKaRa). A plasmid DNA was purified from theresulting Escherichia coli transformant using QIAprep Spin Miniprep Kit(QIAGEN). A nucleotide sequence was analyzed using the resulting plasmidDNA as a template, and using a primer consisting of the nucleotidesequence represented by any of SEQ ID NOs: 29, 49 and 54 according tothe method described in Example 11 (1). As a result, a nucleotidesequence represented by nucleotide numbers 1 to 662 of the nucleotidesequence represented by SEQ ID NO: 42 was read.

Further, a DNA having a nucleotide sequence extending toward to the 3′downstream region from nucleotide number 3534 of the nucleotide sequencerepresented by SEQ ID NO: 42 was cloned. 1.0 μL of CDS-primer attachedto the kit and 1.0 μL of sterilized distilled water were mixed into 3 μL(230 ng) of the total RNA prepared in Example 10 (1), the mixture wasmaintained at 70° C. for 2 minutes, and maintained on ice for 2 minutes.3′ RACE ready cDNA was prepared using the reaction solution as inpreparation of 5′ RACE ready cDNA. PCR amplifying 3′ downstream regionwas performed using this 3′ RACE ready cDNA as a template. A PCRreaction solution was prepared by mixing 5.0 μL of 10× Advantage 2buffer attached to the kit, 1.0 μL of 10 mM dNTP Mix and 1.0 μL of 50×Advantage 2 Polymerase Mix into 2.5 μL of 3′ RACE ready cDNA, adding 5.0μL of 10× Universal Primer A Mix attached to the kit as a primer, and1.0 μL of a 10 μM solution of an oligonucleotide consisting of thenucleotide sequence represented by SEQ ID NO: 42, and adding sterilizeddistilled water to a total amount of 50 μL. This reaction solution wassubjected to repetition of 5 cycles of incubation, each cycle comprisingmaintaining a temperature at 94° C. for 5 seconds, then, at 72° C. for 2minutes, further repetition of 5 cycles of incubation, each cyclecomprising maintaining a temperature at 94° C. for 5 seconds, then, at70° C. for 10 seconds, then, at 72° C. for 2 seconds, further repetitionof 25 cycles of incubation, each cycle comprising maintaining atemperature at 94° C. for 5 seconds, then, at 68° C. for 10 seconds,then, at 72° C. for 2 minutes, followed by maintaining a temperature at72° C. for 7 minutes. The PCR reaction solution and the pCR2.1-TOPOcloning vector (Invitrogen) were ligated to the vector according to theinstruction attached to the kit, after that, which was introduced intoEscherichia coli JM109 (TaKaRa). A plasmid DNA was purified from theresulting Escherichia coli transformant using QIAprep Spin Miniprep Kit(QIAGEN). A nucleotide sequence was analyzed using the resulting plasmidDNA as a template, and using a primer consisting of the nucleotidesequence represented by any of SEQ ID NOs: 29, 50 and 54, according tothe method described in Example 11 (1). As a result, a nucleotidesequence represented by nucleotide numbers 3535 to 3882 of thenucleotide sequence represented by SEQ ID NO: 42 was read.

All analyzed nucleotide sequences were joined and, as a result, thenucleotide sequence represented by SEQ ID NO: 42 was obtained. Thenucleotide sequence represented by SEQ ID NO: 42 consists of 3882 bases(including termination codon), and was a nucleotide sequence encoding1293 amino acid residues (SEQ ID NO: 41). A molecular weight of aprotein having the amino acid sequence represented by SEQ ID NO: 41 wascalculated to be 141818 Da.

(3) Isolation of Full Length Fusarium oxysporum FoOS1 Gene

A DNA having a nucleotide sequence encoding an amino acid sequence ofFusarium oxysporum FoOS1 (hereinafter, referred to as FoOS-1 DNA in somecases) was amplified by PCR using the 5′ RACE ready cDNA prepared inExample 11 (2) as a template. By performing a PCR using, as a primer, anoligonucleotide consisting of the nucleotide sequence represented by SEQID NO: 52 and an oligonucleotide consisting of the nucleotide sequencerepresented by SEQ ID NO: 53, a DNA having the nucleotide sequencerepresented by SEQ ID NO: 42 was amplified. The PCR was performed usingKOD-Plus- (TOYOBO) under the amplifying conditions that a temperaturewas maintained at 94° C. for 2 minutes and, thereafter, 35 cycles ofincubation were repeated, each cycle comprising maintaining atemperature at 94° C. for 15 seconds, then, at 55° C. for 30 seconds,further, at 68° C. for 6 minutes. The PCR reaction solution (50 μL) wasprepared by adding 2.5 μL of 5′ a RACE ready cDNA, 5.0 μL of 10× buffer,5.0 μL of 2 mM dNTPs, 2.0 μL of 25 mM MgSO₄, each 1.0 μL of 10 μMoligonucleotide primers, 32.5 μL of sterilized distilled water and 1.0μL of KOD Plus-. After the reaction, a part of the PCR reaction solutionwas separated by 1% agarose gel electrophoresis, and stained withethidium bromide. It was confirmed that about 4 kb of the DNA (FoOS1DNA) was amplified.

Example 12 Construction of Expression Plasmid of Fusarium oxysporumFoOS1 Gene and Preparation of Transformed Budding Yeast

The FoOS1 DNA was cloned into a pCR2.1-TOPO cloning vector (Invitrogen).About 4 kb of the DNA (FoOS-1 DNA) was purified from the PCR reactionsolution prepared in Example 11 (3) using QIAquick PCR Purification Kit(QIAGEN) according to the instruction attached to the kit. 3′A additionwas performed on about 4 kb of the purified DNA (FoOS-1 DNA) accordingto the method described in Example 11 (1). The 3′A-added about 4 kb DNA(FoOS-1 DNA) and the pCR2.1-TOPO cloning vector (Invitrogen) wereligated according to the manual attached to the cloning vector toconstruct a plasmid pCRFoOS1. A nucleotide sequence of the resultingplasmid was analyzed according to the method described in Example 11(1).As a primer, an oligonucleotide consisting of the nucleotide sequencerepresented by any of SEQ ID NOs: 29, 43 to 51, and 54 was used. As aresult, the nucleotide sequence represented by SEQ ID NO: 42 wasobtained, and it was confirmed that the plasmid pCRFoOS1 was a plasmidcontaining the FoOS-1 DNA.

The FoOS-1 DNA contained in the thus prepared plasmid pCRFoOS1 wascloned into a shuttle vector p415ADH replicable in yeast and Escherichiacoli to construct an expression plasmid. The plasmid pCRFoOS1 wasdigested with restriction enzymes SpeI and PstI and, on the other hand,the shuttle vector p415ADH was also digested with restriction enzymesSpeI and PstI. Each of them was separated by 0.8% agarose gelelectrophoresis, a part of the gel containing the FoOS-1 DNA digestedwith SpeI and PstI and the shuttle vector p415ADH digested with SpeI andPstI was excised, and the FoOS-1 DNA and the shuttle vector wererecovered from the gel using QIAquick Gel Extraction Kit (QIAGEN)according to the attached manual. The FoOS-1 DNA was inserted betweenSpeI site and PstI site in the multicloning site of the shuttle vectorusing Ligation Kit Ver. 2 (TaKaRa) according to the manual attached tothe kit, whereby, an expression plasmid pADHFoOS1 was constructed. Anucleotide sequence of the resulting expression plasmid was analyzedaccording to the method described in Example 11 (1). As a primer, anoligonucleotide consisting of the nucleotide sequence represented by anyof SEQ ID NO: 43 to 53 was used. As a result, the nucleotide sequencerepresented by SEQ ID NO: 42 was obtained, and it was confirmed that theexpression plasmid pADHFoOS1 harbored a DNA having a nucleotide sequenceencoding an amino acid sequence of FoOS-1.

The prepared expression plasmid pADHFoOS1 was introduced into buddingyeast AH22 strain and TM182 strain according to the method described inExample 2. By utilizing the disappearance of leucine auxotrophy in theresulting transformed budding yeast, the transformed budding yeast AH22strain (AH22-FoOS1) was selected on a Glu-Leu agar medium, and thetransformed budding yeast TM182 strain (TM182-FoOS1) was selected on aGal-Ura-Leu agar medium. It was confirmed that the resulting TM182-FoOS1grows even when transplanted to a Glu-Ura-Leu medium.

Example 13 Antifungal Compound Sensitivity Test of Transformed BuddingYeast TM182-FoOS1

The transformed budding yeast AH22-FoOS1 prepared in Example 12 wascultured while shaking at 30° C. for 18 hours in a Glu-Leu medium. As acontrol, the AH22 strain was similarly cultured while shaking at 30° C.for 18 hours in a Glu medium. The absorbance at 600 nm of each growntransformed budding yeast in a cell suspension was measured, and cellsuspension diluted with sterilized distilled water to the absorbance of0.1 was prepared. Further, a cell suspension in which the aforementionedcell suspension of the transformed budding yeast AH22-FoOS1 was diluted50-fold with a Glu-Leu medium, and a cell suspension in which theaforementioned cell suspension of the AH22 strain was diluted 50-foldwith a Glu medium were prepared.

A solution in which each of Compounds (1) to (3) was dissolved indimethylsulfoxide (DMSO) to the concentration of 600 ppm, a solution inwhich each of Compounds (4) and (5) was dissolved in dimethylsulfoxide(DMSO) to the concentration of 2000 ppm, and a solution in whichCompounds (6) and (7) was dissolved in dimethylsulfoxide (DMSO) to theconcentration of 20 ppm were prepared, and two microplates were preparedin which each 1.0 μL per well of the Compound solution and DMSO as acontrol were dispensed. In one microplate among them, each 100 μL ofcell suspensions of the transformed budding yeast AH22-FoOS1 which hadbeen prepared by dilution as described above was dispensed, and culturedby allowing to stand at 30° C. for 26.5 hours. In another microplate,each 100 μL of cell suspensions of the control yeast AH22 strain whichhad been prepared by dilution as described above was dispensed, andcultured by allowing to stand at 30° C. for 24.5 hours. After culturing,the absorbance at 600 nm of each well was measured with a microplatereader.

Similarly, the transformed budding yeast TM182-FoOS1 prepared in Example12 was cultured at 30° C. for 18 hours in a Glu-Ura-Leu medium. Theabsorbance at 600 nm of the grown transformed budding yeast in a cellsuspension was measured, and a cell suspension diluted with sterilizeddistilled water to the absorbance of 0.1 was prepared. Further, a cellsuspension in which the transformed budding yeast TM182-FoOS1 wasdiluted 50-fold with a Glu-Ura-Leu medium and, as a control, a cellsuspension in which the yeast was diluted 50-fold with a Gal-Ura-Leumedium were prepared.

A solution in which each of Compounds (1) to (3) was dissolved indimethylsulfoxide (DMSO) to the concentration of 600 ppm, a solution inwhich each of Compounds (4) and (5) was dissolved in dimethylsulfoxide(DMSO) to the concentration of 2000 ppm, and a solution in which each ofCompounds (6) and (7) was dissolved in dimethylsulfoxide (DMSO) to theconcentration of 20 ppm were prepared, and two microplates were preparedin which each 2.0 μL per well of the Compound-DMSO solution and DMSO asa control were dispensed into 2 wells. In one microplate among them,each 100 μL of cell suspensions of the transformed budding yeastTM182-FoOS1 which had been prepared by dilution with a Glu-Ura-Leumedium as described above was dispensed, and cultured by allowing tostand at 30° C. for 25 hours. In another microplate, as described above,as a control, each 100 μL of cell suspensions of the transformed buddingyeast TM182-FoOS1 which had been prepared by dilution with a Gal-Ura-Leumedium was dispensed, and cultured at 30° C. for 51 hours. Afterculturing, the absorbance at 600 nm of each well was measured with amicroplate reader.

A degree of growth of each transformed budding yeast cultured in thepresence of Compounds (1) to (7) is shown in Table 5. A degree of growthof the transformed budding yeast is expressed as a relative value inpercentage, letting the absorbance at 600 nm at the concentration of theCompound of 0 ppm to be 100. It was confirmed that an inhibiting degreeof growth of the transformed budding yeast TM182-FoOS1 by each testsubstance was grater than an inhibiting degree of growth of thetransformed budding yeast AH22-FoOS1 by each test substance, and thetransformed budding yeast TM182-FoOS1 was a transformed cell with theenhanced sensitivity to an antifungal compound as compared with thetransformed budding yeast AH22-FoOS1.

TABLE 5 Degree of growth of budding yeast AH22- AH22 FoOS1 TM182-FoOS1Test substance Glu Glu-Leu Gal-Ura- Gul-Ura- (final concentration)medium medium Leu medium Leu medium Compoud (1) (6 ppm) 88 81 116  26Compoud (2) (6 ppm) 91 91 87 55 Compoud (3) (6 ppm) 87 86 99 22 Compoud(4) (20 ppm) 96 90 104  20 Compoud (5) (20 ppm) 80 71 80 57 Compoud (6)(0.2 ppm) 92 69 99  7 Compoud (7) (0.2 ppm) 91 88 89 21

Example 14 Isolation of Mycospharella tritici StOS-1 Gene

(1) Analysis of Mycospharella tritici StOS-1 Gene Fragment

The amplified DNA was purified from the reaction solution of PCR whichhad been performed using a primer pair 16 and using a cDNA ofMycospharella tritici as a template in Example 10 (4), using QIAquickPCR Purification Kit (QIAGEN) according to the instruction attached tothe kit. 3′A addition was performed on the purified DNA according to themethod described in Example 11 (1). The 3′A-added DNA and thepCR2.1-TOPO cloning vector (Invitrogen) were ligated according to theinstruction attached to the cloning vector, and this was introduced intoEscherichia coli JM109 (TaKaRa).

DNA was purified from the resulting Escherichia coli transformant bycolony PCR using Ex Taq HS (TaKaRa). The PCR reaction solution (15 μL)was prepared by mixing 1.5 μL of 10× buffer, 2.25 μL of 10 mM dNTPs,0.15 μL of Ex Taq HS, each 0.4 μL of a 10 μM solution of anoligonucleotide consisting of the nucleotide sequence represented by SEQID NO: 66 and a 10 μM solution of an oligonucleotide consisting of thenucleotide sequence represented by SEQ ID NO: 67, and 10.3 μL ofsterilized distilled water, and adding a part of the Escherichia colitransformant colony thereto. PCR was performed under the amplifyingconditions that this reaction solution was maintained at 97° C. for 2minutes and, thereafter, 35 cycles of incubation were repeated, eachcycle comprising maintaining a temperature at 97° C. for 15 seconds,then, at 55° C. for 15 seconds, then, at 72° C. for 3 minutes. Theamplified DNA was purified from the PCR reaction solution aftertemperature maintenance using QIAquick PCR Purification Kit (QIAGEN)according to the instruction attached to the kit. A nucleotide sequencewas analyzed using oligonucleotides consisting of nucleotide sequencesrepresented by SEQ ID NOs: 29 and 54 as a primer and employing thepurified DNA as a template according to the method described in Example11 (1). As a result, a nucleotide sequence represented by base numbers2241 to 3603 of the nucleotide sequence represented by SEQ ID NO: 56 wasread.

(2) Analysis of Full Length Mycospharella tritici StOS-1 Gene

A DNA having a nucleotide sequence extending toward to 5′ upstreamregion of a base number 2241 of the nucleotide sequence represented bySEQ ID NO: 56 was cloned using SMART RACE cDNA Amplification Kit(CLONTECH) according to the instruction attached to the kit. A reactionsolution was prepared by mixing 1.0 μL of CDS-primer and 1.0 μL of SMARTIIA Oligo attached to the kit into 3 μL (230 ng) of total RNA preparedin Example 10 (2), a temperature was maintained at 70° C. for 2 minutes,and maintained on ice for 2 minutes. To the reaction solution were added2 μL of 5× First-Strand buffer attached to the kit, 1 μL of 20 mM DTT, 1μL of 10 mM dNTP Mix and 1 μL of PowerScript Reverse Transcriptase, tomix them, and the mixture was maintained at 42° C. for 1.5 hours. To thereaction solution after temperature maintenance was added 100 μL ofTricine-EDTA buffer attached to the kit, a temperature was maintained at72° C. for 7 minutes, thus 5′ RACE ready cDNA was prepared. PCRamplifying 5′ upstream region was performed using this 5′ RACE readycDNA as a template and using KOD-plus- (TOYOBO). The PCR reactionsolution was prepared by mixing 2.5 μL of 5′ RACE ready cDNA, 5.0 μL of10× buffer, 5.0 μL of 2 mM dNTPs, 2.0 μL of 25 mM MgSO₄ and 1.0 μL ofKOD-Plus, adding 5.0 μL of 10× Universal Primer A Mix attached to thekit and 1.0 μL of a 10 μM solution of an oligonucleotide consisting ofthe nucleotide sequence represented by SEQ ID NO: 43 as primers, andadding sterilized distilled water to a total amount of 50 μL. Thisreaction solution was maintained at 94° C. for 2 minutes, and 35 cyclesof incubation were repeated, each cycle comprising maintaining atemperature at 94° C. for 15 seconds, then, at 68° C. for 5 minutes. Theamplified DNA was purified from the PCR reaction solution using QIAquickPCR Purification Kit (QIAGEN) according to the instruction attached tothe kit, and then, 3′A addition was performed on the DNA according tothe method described in Example 11(1). The 3′A-added DNA and thepCR2.1-TOPO cloning vector (Invitrogen) were ligated according to theinstruction attached to the cloning vector, after that, which wasintroduced into Escherichia coli JM109 (TaKaRa). A plasmid DNA waspurified from the resulting Escherichia coli transformant using QIAprepSpin Miniprep Kit (QIAGEN). A nucleotide sequence was analyzed using theresulting plasmid DNA as a template and using primers consisting ofnucleotide sequences represented by SEQ ID NOs: 29, 54, and 59 to 61according to the method described in Example 11(1). As a result, anucleotide sequence represented by base numbers 1 to 2240 of thenucleotide sequence represented by SEQ ID NO: 56 was read.

Further, a DNA having a nucleotide sequence extending toward to the 3′downstream region from nucleotide number 3603 of the nucleotide sequencerepresented by SEQ ID NO: 56 was cloned. 1.0 μL of CDS-primer attachedto the kit and 1.0 μL of sterilized distilled water were mixed into 3 μL(230 ng) of the total RNA prepared in Example 10 (2), the mixture wasmaintained at 70° C. for 2 minutes, and maintained on ice for 2 minutes.3′ RACE ready cDNA was prepared using the reaction solution as inpreparation of 5′ RACE ready cDNA. PCR amplifying 3′ downstream regionwas performed using this 3′ RACE ready cDNA as a template. A PCRreaction solution was prepared by mixing 5.0 μL of 10× Advantage 2buffer attached to the kit, 1.0 μL of 10 mM dNTP Mix and 1.0 μL of 50×Advantage 2 Polymerase Mix into 2.5 μL of 3′ RACE ready cDNA, adding 5.0μL of 10× Universal Primer A Mix attached to the kit as a primer, and1.0 μL of a 10 μM solution of an oligonucleotide consisting of thenucleotide sequence represented by SEQ ID NO: 58, and adding sterilizeddistilled water to a total amount of 50 μL. This reaction solution wassubjected to repetition of 5 cycles of incubation, each cycle comprisingmaintaining a temperature at 94° C. for 5 seconds, then, at 72° C. for 4minutes, further repetition of 5 cycles of incubation, each cyclecomprising maintaining a temperature at 94° C. for 5 seconds, then, at70° C. for 10 seconds, then, at 72° C. for 4 minutes, further repetitionof 25 cycles of incubation, each cycle comprising maintaining atemperature at 94° C. for 5 seconds, then, at 68° C. for 10 seconds,then, at 72° C. for 4 minutes, followed by maintaining a temperature at72° C. for 7 minutes. The PCR reaction solution and the pCR2.1-TOPOcloning vector (Invitrogen) were ligated to the vector according to theinstruction attached to the kit, after that, which was introduced intoEscherichia coli JM109 (TaKaRa). A plasmid DNA was purified from theresulting Escherichia coli transformant using QIAprep Spin Miniprep Kit(QIAGEN). A nucleotide sequence was analyzed using the resulting plasmidDNA as a template, and using a primer consisting of the nucleotidesequence represented by any of SEQ ID NOs: 29 and 54, according to themethod described in Example 11 (1). As a result, a nucleotide sequencerepresented by nucleotide numbers 3604 to 3924 of the nucleotidesequence represented by SEQ ID NO: 56 was read.

All analyzed nucleotide sequences were joined and, as a result, thenucleotide sequence represented by SEQ ID NO: 56 was obtained. Thenucleotide sequence represented by SEQ ID NO: 56 consists of 3924 bases(including termination codon), and was a nucleotide sequence encoding1307 amino acid residues (SEQ ID NO: 55). A molecular weight of aprotein having the amino acid sequence represented by SEQ ID NO: 55 wascalculated to be 143276 Da.

(3) Isolation of Full Length Mycospharella tritici StOS-1 Gene

A DNA having a nucleotide sequence encoding an amino acid sequence ofMycospharella tritici StOS-1 (hereinafter, referred to as StOS-1 DNA insome cases) was amplified by PCR using the 5′ RACE ready cDNA preparedin Example 14 (2) as a template. By performing a PCR using, as a primer,an oligonucleotide consisting of the nucleotide sequence represented bySEQ ID NO: 64 and an oligonucleotide consisting of the nucleotidesequence represented by SEQ ID NO: 65, a DNA having the nucleotidesequence represented by SEQ ID NO: 56 was amplified, according to themethod described in Example 11 (3). After the reaction, a part of thePCR reaction solution was separated by 1% agarose gel electrophoresis,and stained with ethidium bromide. It was confirmed that about 4 kb ofthe DNA (StOS-1 DNA) was amplified.

Example 15 Construction of Expression Plasmid of Mycospharella triticiStOS-1 Gene and Preparation of Transformed Budding Yeast

The StOS-1 DNA was cloned into a pCR2.1-TOPO cloning vector(Invitrogen). About 4 kb of the DNA (StOS-1 DNA) was purified from thePCR reaction solution prepared in Example 14 (3) using QIAquick PCRPurification Kit (QIAGEN) according to the instruction attached to thekit. 3′A addition was performed on about 4 kb of the purified DNA(StOS-1 DNA) according to the method described in Example 11 (1). The3′A-added about 4 kb DNA (StOS-1 DNA) and the pCR2.1-TOPO cloning vector(Invitrogen) were ligated according to the manual attached to thecloning vector to construct a plasmid pCRStOS1. A nucleotide sequence ofthe resulting plasmid was analyzed according to the method described inExample 11(1). As a primer, an oligonucleotide consisting of thenucleotide sequence represented by any of SEQ ID NOs: 29, 54, and 58 to63 was used. As a result, the nucleotide sequence represented by SEQ IDNO: 56 was obtained, and it was confirmed that the plasmid pCRStOS1 wasa plasmid containing the StOS-1 DNA.

The StOS-1 DNA contained in the thus prepared plasmid pCRStOS1 wascloned into a shuttle vector p415ADH replicable in yeast and Escherichiacoli to construct an expression plasmid. The plasmid pCRStOS1 wasdigested with restriction enzymes SpeI and HindIII and, on the otherhand, the shuttle vector p415ADH was also digested with restrictionenzymes SpeI and HindIII. Each of them was separated by 0.8% agarose gelelectrophoresis, a part of the gel containing the StOS-1 DNA digestedwith SpeI and HindIII and the shuttle vector p415ADH digested with SpeIand HindIII was excised, and the StOS-1 DNA and the shuttle vector wererecovered from the gel using QIAquick Gel Extraction Kit (QIAGEN)according to the attached manual. The StOS-1 DNA was inserted betweenSpeI site and HindIII site in the multicloning site of the shuttlevector using Ligation Kit Ver. 2 (TaKaRa) according to the manualattached to the kit, whereby, an expression plasmid pADHStOS1 wasconstructed. A nucleotide sequence of the resulting expression plasmidwas analyzed according to the method described in Example 11 (1). As aprimer, an oligonucleotide consisting of the nucleotide sequencerepresented by any of SEQ ID NO: 58 to 65 was used. As a result, thenucleotide sequence represented by SEQ ID NO: 56 was obtained, and itwas confirmed that the expression plasmid pADHStOS1 harbored a DNAhaving a nucleotide sequence encoding an amino acid sequence of StOS-1.

The prepared expression plasmid pADHStOS1 was introduced into buddingyeast AH22 strain and TM182 strain according to the method described inExample 2. By utilizing the disappearance of leucine auxotrophy in theresulting transformed budding yeast, the transformed budding yeast AH22strain (AH22-StOS1) was selected on a Glu-Leu agar medium, and thetransformed budding yeast TM182 strain (TM182-StOS1) was selected on aGal-Ura-Leu agar medium. It was confirmed that the resulting TM182-StOS1grows even when transplanted to a Glu-Ura-Leu medium.

Example 16 Antifungal Compound Sensitivity Test of Transformed BuddingYeast TM182-StOS1

The transformed budding yeast AH22-StOS1 prepared in Example 15 wascultured while shaking at 30° C. for 18 hours in a Glu-Leu medium. As acontrol, the AH22 strain was similarly cultured while shaking at 30° C.for 18 hours in a Glu medium. The absorbance at 600 nm of each growntransformed budding yeast in a cell suspension was measured, and cellsuspension diluted with sterilized distilled water to the absorbance of0.1 was prepared. Further, a cell suspension in which the aforementionedcell suspension of the transformed budding yeast AH22-StOS1 was diluted50-fold with a Glu-Leu medium, and a cell suspension in which theaforementioned cell suspension of the AH22 strain was diluted 50-foldwith a Glu medium were prepared.

A solution in which each of Compounds (1) to (3) was dissolved indimethylsulfoxide (DMSO) to the concentration of 6 ppm, a solution inwhich each of Compounds (4) and (5) was dissolved in dimethylsulfoxide(DMSO) to the concentration of 2000 ppm, and a solution in whichCompounds (6) and (7) was dissolved in dimethylsulfoxide (DMSO) to theconcentration of 20 ppm were prepared, and two microplates were preparedin which each 1.0 μL per well of the Compound solution and DMSO as acontrol were dispensed. In one microplate among them, each 100 μL ofcell suspensions of the transformed budding yeast AH22-StOS1 which hadbeen prepared by dilution as described above was dispensed, and culturedby allowing to stand at 30° C. for 28 hours. In another microplate, each100 μL of cell suspensions of the control yeast AH22 strain which hadbeen prepared by dilution as described above was dispensed, and culturedby allowing to stand at 30° C. for 24.5 hours. After culturing, theabsorbance at 600 nm of each well was measured with a microplate reader.

Similarly, the transformed budding yeast TM182-StOS1 prepared in Example15 was cultured at 30° C. for 18 hours in a Glu-Ura-Leu medium. Theabsorbance at 600 nm of the grown transformed budding yeast in a cellsuspension was measured, and a cell suspension diluted with sterilizeddistilled water to the absorbance of 0.1 was prepared. Further, a cellsuspension in which the transformed budding yeast TM182-StOS1 wasdiluted 50-fold with a Glu-Ura-Leu medium and, as a control, a cellsuspension in which the yeast was diluted 50-fold with a Gal-Ura-Leumedium were prepared.

A solution in which each of Compounds (1) to (3) was dissolved indimethylsulfoxide (DMSO) to the concentration of 6 ppm, a solution inwhich each of Compounds (4) and (5) was dissolved in dimethylsulfoxide(DMSO) to the concentration of 2000 ppm, and a solution in which each ofCompounds (6) and (7) was dissolved in dimethylsulfoxide (DMSO) to theconcentration of 20 ppm were prepared, and two microplates were preparedin which each 2.0 μL per well of the Compound-DMSO solution and DMSO asa control were dispensed into 2 wells. In one microplate among them,each 100 μL of cell suspensions of the transformed budding yeastTM182-StOS1 which had been prepared by dilution with a Glu-Ura-Leumedium as described above was dispensed, and cultured by allowing tostand at 30° C. for 26.5 hours. In another microplate, as describedabove, as a control, each 100 μL of cell suspensions of the transformedbudding yeast TM182-StOS1 which had been prepared by dilution with aGal-Ura-Leu medium was dispensed, and cultured at 30° C. for 49.5 hours.After culturing, the absorbance at 600 nm of each well was measured witha microplate reader.

A degree of growth of each transformed budding yeast cultured in thepresence of Compounds (1) to (7) is shown in Table 6. A degree of growthof the transformed budding yeast is expressed as a relative value inpercentage, letting the absorbance at 600 nm at the concentration of theCompound of 0 ppm to be 100. It was confirmed that an inhibiting degreeof growth of the transformed budding yeast TM182-StOS1 by each testsubstance was grater than an inhibiting degree of growth of thetransformed budding yeast AH22-StOS1 by each test substance, and thetransformed budding yeast TM182-StOS1 was a transformed cell with theenhanced sensitivity to an antifungal compound as compared with thetransformed budding yeast AH22-StOS1.

TABLE 6 Degree of growth of budding yeast AH22- AH22 StOS1 TM182-StOS1Test substance Glu Glu-Leu Gal-Ura- Gul-Ura- (final concentration)medium medium Leu medium Leu medium Compoud (1) (0.6 ppm) 99 101  101 67  Compoud (2) (0.6 ppm) 94 100  97 23  Compoud (3) (0.6 ppm) 96 98 9419  Compoud (4) (20 ppm) 96 91 99 7 Compoud (5) (20 ppm) 80 76 74 6Compoud (6) (0.2 ppm) 92 93 97 6 Compoud (7) (0.2 ppm) 91 91 91 9

Example 17 Isolation of Thanatephorus cucumeris RsOS-1 Gene

(1) Analysis of Thanatephorus cucumeris RsOS-1 Gene Fragment

The amplified DNA was purified from the reaction solution of PCR whichhad been performed using a primer pair 3 and using a cDNA ofThanatephorus cucumeris as a template in Example 10 (5), using QIAquickPCR Purification Kit (QIAGEN) according to the instruction attached tothe kit. 3′A addition was performed on the purified DNA according to themethod described in Example 11 (1). The 3′A-added DNA and thepCR2.1-TOPO cloning vector (Invitrogen) were ligated according to theinstruction attached to the cloning vector, and this was introduced intoEscherichia coli JM109 (TaKaRa).

DNA was purified from the resulting Escherichia coli transformant bycolony PCR using Ex Taq HS (TaKaRa). The PCR reaction solution (15 μL)was prepared by mixing 1.5 μL of 10× buffer, 2.25 μL of 10 mM dNTPs,0.15 μL of Ex Taq HS, each 0.4 μL of a 10 μM solution of anoligonucleotide consisting of the nucleotide sequence represented by SEQID NO: 28 and a 10 μM solution of an oligonucleotide consisting of thenucleotide sequence represented by SEQ ID NO: 29, and 10.3 μL ofsterilized distilled water, and adding a part of the Escherichia colitransformant colony thereto. PCR was performed under the amplifyingconditions that this reaction solution was maintained at 97° C. for 2minutes and, thereafter, 35 cycles of incubation were repeated, eachcycle comprising maintaining a temperature at 97° C. for 15 seconds,then, at 55° C. for 15 seconds, then, at 72° C. for 3 minutes. Theamplified DNA was purified from the PCR reaction solution aftertemperature maintenance using QIAquick PCR Purification Kit (QIAGEN)according to the instruction attached to the kit. A nucleotide sequencewas analyzed using oligonucleotides consisting of nucleotide sequencesrepresented by SEQ ID NOs: 28 and 29 as a primer and employing thepurified DNA as a template according to the method described in Example11 (1). As a result, a nucleotide sequence represented by base numbers2838 to 3165 of the nucleotide sequence represented by SEQ ID NO: 69 wasread.

(2) Analysis of Full Length Thanatephorus cucumeris RsOs-1 Gene

A DNA having a nucleotide sequence extending toward to 3′ downstreamregion of a base number 3165 of the nucleotide sequence represented bySEQ ID NO: 69 was cloned using SMART RACE cDNA Amplification Kit(CLONTECH) according to the instruction attached to the kit. A reactionsolution was prepared by mixing 1.0 μL of CDS-primer and 1.0 μL ofsterilized distilled water attached to the kit into 3 μL (253 ng) oftotal RNA prepared in Example 10 (3), a temperature was maintained at70° C. for 2 minutes, and maintained on ice for 2 minutes. To thereaction solution were added 2 μL of 5× First-Strand buffer attached tothe kit, 1 μL of 20 mM DTT, 1 μL of 10 mM dNTP Mix and 1 μL ofPowerScript Reverse Transcriptase, to mix them, and the mixture wasmaintained at 42° C. for 1.5 hours. To the reaction solution aftertemperature maintenance was added 100 μL of Tricine-EDTA buffer attachedto the kit, a temperature was maintained at 72° C. for 7 minutes, thus3′ RACE ready cDNA was prepared. PCR amplifying 3′ downstream region wasperformed using this 3′ RACE ready cDNA as a template and usingKOD-plus- (TOYOBO). The PCR reaction solution was prepared by mixing 2.5μL of 3′ RACE ready cDNA, 5.0 μL of 10× buffer, 5.0 μL of 2 mM dNTPs,2.0 μL of 25 mM MgSO₄ and 1.0 μL of KOD-Plus, adding 5.0 μL of 10×Universal Primer A Mix attached to the kit and 1.0 μL of a 10 μMsolution of an oligonucleotide consisting of the nucleotide sequencerepresented by SEQ ID NO: 70 as primers, and adding sterilized distilledwater to a total amount of 50 μL. This reaction solution was maintainedat 94° C. for 2 minutes, and 35 cycles of incubation were repeated, eachcycle comprising maintaining a temperature at 94° C. for 15 seconds,then, at 68° C. for 6 minutes. The amplified DNA was purified from thePCR reaction solution using QIAquick PCR Purification Kit (QIAGEN)according to the instruction attached to the kit, and then, 3′A additionwas performed on the DNA according to the method described in Example11(1). The 3′A-added DNA and the pCR2.1-TOPO cloning vector (Invitrogen)were ligated according to the instruction attached to the cloningvector, after that, which was introduced into Escherichia coli JM109(TaKaRa). A plasmid DNA was purified from the resulting Escherichia colitransformant using QIAprep Spin Miniprep Kit (QIAGEN). A nucleotidesequence was analyzed using the resulting plasmid DNA as a template andusing primers consisting of nucleotide sequences represented by SEQ IDNOs: 28, 29, and 73 to 76 according to the method described in Example11(1). As a result, a nucleotide sequence represented by base numbers3119 to 4317 of the nucleotide sequence represented by SEQ ID NO: 69 wasread.

Further, a DNA having a nucleotide sequence extending toward to the 5′upstream region from nucleotide number 2838 of the nucleotide sequencerepresented by SEQ ID NO: 69 was cloned. 1.0 μL of CDS-primer attachedto the kit and 1.0 μL of SMART IIA Oligo were mixed into 3 μL (253 ng)of the total RNA prepared in Example 10 (3), the mixture was maintainedat 70° C. for 2 minutes, and maintained on ice for 2 minutes. 5′ RACEready cDNA was prepared using the reaction solution as in preparation of3′ RACE ready cDNA. PCR amplifying 5′ upstream region was performedusing this 5′ RACE ready cDNA as a template and using KOD-plus-(TOYOBO). The PCR reaction solution was prepared by mixing 2.5 μL of 5′RACE ready cDNA, 5.0 μL of 10× buffer, 5.0 μL of 2 mM dNTPs, 2.0 μL of25 mM MgSO₄ and 1.0 μL of KOD-Plus, adding 5.0 μL of 10× UniversalPrimer A Mix attached to the kit and 1.0 μL of a 10 μM solution of anoligonucleotide consisting of the nucleotide sequence represented by SEQID NO: 71 as primers, and adding sterilized distilled water to a totalamount of 50 μL. This reaction solution was maintained at 94° C. for 2minutes, and 35 cycles of incubation were repeated, each cyclecomprising maintaining a temperature at 94° C. for 15 seconds, then, at68° C. for 6 minutes. Using the resulting PCR reaction solution as atemplate, the PCR reaction solution for a further PCR was prepared byadding 5.0 μL of 10× buffer, 5.0 μL of 2 mM dNTPs, 2.0 μL of 25 mM MgSO₄and 1.0 μL of KOD-Plus, 1.0 μL of 10 μM Nested universal primer attachedto the kit and 1.0 μL of a 10 μM solution of an oligonucleotideconsisting of the nucleotide sequence represented by SEQ ID NO: 72 asprimers, and adding sterilized distilled water to a total amount of 50μL. This reaction solution was maintained at 94° C. for 2 minutes, and20 cycles of incubation were repeated, each cycle comprising maintaininga temperature at 94° C. for 15 seconds, then, at 68° C. for 6 minutes.The PCR reaction solution and the pCR2.1-TOPO cloning vector(Invitrogen) were ligated to the vector according to the instructionattached to the kit, after that, which was introduced into Escherichiacoli JM109 (TaKaRa). A plasmid DNA was purified from the resultingEscherichia coli transformant using QIAprep Spin Miniprep Kit (QIAGEN).A nucleotide sequence was analyzed using the resulting plasmid DNA as atemplate, and using a primer consisting of the nucleotide sequencerepresented by any of SEQ ID NOs: 28, 29, and 77 to 82, according to themethod described in Example 11 (1). As a result, a nucleotide sequencerepresented by nucleotide numbers 1 to 3042 of the nucleotide sequencerepresented by SEQ ID NO: 69 was read.

All analyzed nucleotide sequences were joined and, as a result, thenucleotide sequence represented by SEQ ID NO: 69 was obtained. Thenucleotide sequence represented by SEQ ID NO: 69 consists of 4317 bases(including termination codon), and was a nucleotide sequence encoding1438 amino acid residues (SEQ ID NO: 68). A molecular weight of aprotein having the amino acid sequence represented by SEQ ID NO: 68 wascalculated to be 155296 Da.

(3) Isolation of Full Length Thanatephorus cucumeris RsOS-1 Gene

A DNA having a nucleotide sequence encoding an amino acid sequence ofThanatephorus cucumeris RsOs-1 (hereinafter, referred to as RsOS-1 DNAin some cases) was amplified by PCR using a cDNA of Thanatephoruscucumeris prepared in Example 10 (5) as a template. By performing a PCRusing, as a primer, an oligonucleotide consisting of the nucleotidesequence represented by SEQ ID NO: 85 and an oligonucleotide consistingof the nucleotide sequence represented by SEQ ID NO: 86, a DNA havingthe nucleotide sequence represented by SEQ ID NO: 69 was amplified,according to the method described in Example 11 (3). After the reaction,a part of the PCR reaction solution was separated by 1% agarose gelelectrophoresis, and stained with ethidium bromide. It was confirmedthat about 4 kb of the DNA (RsOS-1 DNA) was amplified.

Example 18 Construction of Expression Plasmid of Thanatephorus cucumerisRsOS-1 Gene and Preparation of Transformed Budding Yeast

The RsOS-1 DNA was cloned into a pCR2.1-TOPO cloning vector(Invitrogen). About 4 kb of the DNA (RsOS-1 DNA) was purified from thePCR reaction solution prepared in Example 17 (3) using QIAquick PCRPurification Kit (QIAGEN) according to the instruction attached to thekit. 3′A addition was performed on about 4 kb of the purified DNA(StOS-1 DNA) according to the method described in Example 11 (1). The3′A-added about 4 kb DNA (RsOS-1 DNA) and the pCR2.1-TOPO cloning vector(Invitrogen) were ligated according to the manual attached to thecloning vector to construct a plasmid pCRRsOS1. A nucleotide sequence ofthe resulting plasmid was analyzed according to the method described inExample 11(1). As a primer, an oligonucleotide consisting of thenucleotide sequence represented by any of SEQ ID NOs: 28, 29, 70 to 73,75, 77, 78, and 81 to 84 was used. As a result, the nucleotide sequencerepresented by SEQ ID NO: 69 was obtained, and it was confirmed that theplasmid pCRRsOS1 was a plasmid containing the RsOS-1 DNA.

The RsOS-1 DNA contained in the thus prepared plasmid pCRRsOS1 wascloned into a shuttle vector p415ADH replicable in yeast and Escherichiacoli to construct an expression plasmid. The plasmid pCRRsOS1 wasdigested with restriction enzymes SpeI and HindIII and, on the otherhand, the shuttle vector p415ADH was also digested with restrictionenzymes SpeI and HindIII. Each of them was separated by 0.8% agarose gelelectrophoresis, a part of the gel containing the RsOS-1 DNA digestedwith SpeI and HindIII and the shuttle vector p415ADH digested with SpeIand HindIII was excised, and the RsOS-1 DNA and the shuttle vector wererecovered from the gel using QIAquick Gel Extraction Kit (QIAGEN)according to the attached manual. The RsOS-1 DNA was inserted betweenSpeI site and HindIII site in the multicloning site of the shuttlevector using Ligation Kit Ver. 2 (TaKaRa) according to the manualattached to the kit, whereby, an expression plasmid pADHRsOS1 wasconstructed. A nucleotide sequence of the resulting expression plasmidwas analyzed according to the method described in Example 11 (1). As aprimer, an oligonucleotide consisting of the nucleotide sequencerepresented by any of SEQ ID NO: 70 to 73, 75, 77, 78, 81 to 84, 87 and88 was used. As a result, the nucleotide sequence represented by SEQ IDNO: 69 was obtained, and it was confirmed that the expression plasmidpADHRsOS1 harbored a DNA having a nucleotide sequence encoding an aminoacid sequence of RsOS-1.

The prepared expression plasmid pADHRsOS1 was introduced into buddingyeast AH22 strain and TM182 strain according to the method described inExample 2. By utilizing the disappearance of leucine auxotrophy in theresulting transformed budding yeast, the transformed budding yeast AH22strain (AH22-RsOS1) was selected on a Glu-Leu agar medium, and thetransformed budding yeast TM182 strain (TM182-RsOS1) was selected on aGal-Ura-Leu agar medium. It was confirmed that the resulting TM182-RsOS1grows even when transplanted to a Glu-Ura-Leu medium.

Example 19 Antifungal Compound Sensitivity Test of Transformed BuddingYeast TM182-RsOS1

The transformed budding yeast AH22-RsOS1 prepared in Example 18 wascultured while shaking at 30° C. for 18 hours in a Glu-Leu medium. As acontrol, the AH22 strain was similarly cultured while shaking at 30° C.for 18 hours in a Glu medium. The absorbance at 600 nm of each growntransformed budding yeast in a cell suspension was measured, and cellsuspension diluted with sterilized distilled water to the absorbance of0.1 was prepared. Further, a cell suspension in which the aforementionedcell suspension of the transformed budding yeast AH22-RsOS1 was diluted50-fold with a Glu-Leu medium, and a cell suspension in which theaforementioned cell suspension of the AH22 strain was diluted 50-foldwith a Glu medium were prepared.

A solution in which each of Compounds (1) to (5) was dissolved indimethylsulfoxide (DMSO) to the concentration of 600 ppm, and a solutionin which Compounds (6) and (7) was dissolved in dimethylsulfoxide (DMSO)to the concentration of 60 ppm were prepared, and two microplates wereprepared in which each 1.0 μL per well of the Compound solution and DMSOas a control were dispensed. In one microplate among them, each 100 μLof cell suspensions of the transformed budding yeast AH22-RsOS1 whichhad been prepared by dilution as described above was dispensed, andcultured by allowing to stand at 30° C. for 29.8 hours. In anothermicroplate, each 100 μL of cell suspensions of the control yeast AH22strain which had been prepared by dilution as described above wasdispensed, and cultured by allowing to stand at 30° C. for 24.8 hours.After culturing, the absorbance at 600 nm of each well was measured witha microplate reader.

Similarly, the transformed budding yeast TM182-RsOS1 prepared in Example18 was cultured at 30° C. for 18 hours in a Glu-Ura-Leu medium. Theabsorbance at 600 nm of the grown transformed budding yeast in a cellsuspension was measured, and a cell suspension diluted with sterilizeddistilled water to the absorbance of 0.1 was prepared. Further, a cellsuspension in which the transformed budding yeast TM182-RsOS1 wasdiluted 50-fold with a Glu-Ura-Leu medium. As a control, the transformedbudding yeast TM182-RsOS1 was cultured at 30° C. for 18 hours in aGal-Ura-Leu medium. The absorbance at 600 nm of the grown transformedbudding yeast in a cell suspension was measured, and a cell suspensiondiluted with sterilized distilled water to the absorbance of 0.1 wasprepared. Further, a cell suspension in which the transformed buddingyeast TM182-RsOS1 was diluted 50-fold with a Gal-Ura-Leu medium.

A solution in which each of Compounds (1) to (5) was dissolved indimethylsulfoxide (DMSO) to the concentration of 600 ppm, and a solutionin which each of Compounds (6) and (7) was dissolved indimethylsulfoxide (DMSO) to the concentration of 60 ppm were prepared,and two microplates were prepared in which each 2.0 μL per well of theCompound-DMSO solution and DMSO as a control were dispensed into 2wells. In one microplate among them, each 100 μL of cell suspensions ofthe transformed budding yeast TM182-RsOS1 which had been prepared bydilution with a Glu-Ura-Leu medium as described above was dispensed, andcultured by allowing to stand at 30° C. for 26.8 hours. In anothermicroplate, as described above, as a control, each 100 μL of cellsuspensions of the transformed budding yeast TM182-RsOS1 which had beenprepared by dilution with a Gal-Ura-Leu medium was dispensed, andcultured at 30° C. for 42.5 hours. After culturing, the absorbance at600 nm of each well was measured with a microplate reader.

A degree of growth of each transformed budding yeast cultured in thepresence of Compounds (1) to (7) is shown in Table 7. A degree of growthof the transformed budding yeast is expressed as a relative value inpercentage, letting the absorbance at 600 nm at the concentration of theCompound of 0 ppm to be 100. It was confirmed that an inhibiting degreeof growth of the transformed budding yeast TM182-RsOS1 by each testsubstance was grater than an inhibiting degree of growth of thetransformed budding yeast AH22-RsOS1 by each test substance, and thetransformed budding yeast TM182-RsOS1 was a transformed cell with theenhanced sensitivity to an antifungal compound as compared with thetransformed budding yeast AH22-RsOS1.

TABLE 7 Degree of growth of budding yeast AH22- AH22 RsOS1 TM182-RsOS1Test substance Glu Glu-Leu Gal-Ura- Gul-Ura- (final concentration)medium medium Leu medium Leu medium Compoud (1) (6.0 ppm) 88 103 108 15Compoud (2) (6.0 ppm) 92 101  96 11 Compoud (3) (6.0 ppm) 82 101 101 27Compoud (4) (6.0 ppm) 83  89  88 17 Compoud (5) (6.0 ppm) 78  85 101  9Compoud (6) (0.6 ppm) 79  79 100 12 Compoud (7) (0.6 ppm) 85 101  99 31

Example 20 Isolation of a Gene of the Present Histidine Kinase ofPhytophthora infestans (Hereinafter, Referred to PiOS-1 Gene)

(1) Analysis of Phytophthora infestans PiOS-1 Gene Fragment

The amplified DNA was purified from the reaction solution of PCR whichhad been performed using a primer pair 6 and using a cDNA ofPhytophthora infestans as a template in Example 10(5), using QIAquickPCR Purification Kit (QIAGEN) according to the instruction attached tothe kit. 3′A addition was performed on the purified DNA according to themethod described in Example 11 (1). The 3′A added DNA and thepCR2.1-TOPO cloning vector (Invitrogen) were ligated according to theinstruction attached to the cloning vector, after that, which wasintroduced into Escherichia coli JM109 (TaKaRa).

A DNA was amplified from the resulting Escherichia coli transformant bycolony PCR using Ex Taq HS (TaKaRa). The PCR reaction solution (15 μL)was prepared by mixing 1.5 μL of 10× buffer, 2.25 μL of 10 mM dNTPs,0.15 μL of Ex Taq HS, each 0.4 μL of a 10 μM solution of anoligonucleotide consisting of the nucleotide sequence represented by SEQID NO: 28 and a 10 μM solution of an oligonucleotide consisting of thenucleotide sequence represented by SEQ ID NO: 29, and 10.3 μL ofsterilized distilled water, and adding a part of the Escherichia colitransformant colony thereto. PCR was performed under the amplifyingconditions that this reaction solution was maintained at 97° C. for 2minutes, and 35 cycles of incubation were repeated, each cyclecomprising maintaining a temperature at 97° C. for 15 seconds, then, at55° C. for 15 seconds, further, at 72° C. for 3 minutes. The amplifiedDNA was purified from the PCR reaction solution after temperaturemaintenance using QIAquick PCR purification Kit (QIAGEN) according tothe manual attached to the kit. A nucleotide sequence was analyzed usingthe purified DNA as a template and using oligonucleotides consisting ofthe nucleotide sequence represented by any of SEQ ID NOs: 28 and 29 asprimers according to the method described in Example 11(1). As a result,a nucleotide sequence represented by SEQ ID NO: 89 containing anucleotide sequence of an oligonucleotide used as a primer pair 6 wasread.

(2) Analysis of Full Length Phytophthora infestans PiOS-1 Gene

A DNA having a nucleotide sequence extending toward to 5′ upstreamregion of a nucleotide sequence represented by SEQ ID NO: 89 is clonedusing SMART RACE cDNA Amplification Kit (CLONTECH) according to theinstruction attached to the kit. A reaction solution is prepared bymixing 1.0 μL of CDS-primer attached to the kit and 1.0 μL of SMART IIAOligo into 3 μL (200 ng) of the total RNA prepared in Example 10 (4), atemperature is maintained at 70° C. for 2 minutes, and is maintained onice for 2 minutes. To the reaction solution are added 2 μL of 5×First-Strand buffer attached to the kit, 1 μL of 20 mM DTT, 1 μL of 10mM dNTP Mix and 1 μL of PowerScript Reverse Transcriptase to mix them,and the mixture is maintained at 42° C. for 1.5 hours. To the reactionsolution after temperature maintenance is added 100 μL of Tricine-EDTAbuffer attached to the kit, a temperature is maintained at 72° C. for 7minutes, and 5′ RACE ready cDNA is prepared. PCR amplifying 5′ upstreamregion is performed using this 5′ RACE ready cDNA as a template andusing KOD-plus- (TOYOBO). The PCR reaction solution is prepared bymixing 2.5 μL of 5′ RACE ready cDNA, 5.0 μL of 10× buffer, 5.0 μL of 2mM dNTPs, 2.0 μL of 25 mM MgSO₄ and 1.0 μL of KOD-Plus-, adding 5.0 μLof 10× Universal primer A Mix attached to the kit as a primer and 1.0 μLof a 10 μM solution of an oligonucleotide consisting of 20 to 30 basesselected from complementary sequences of the nucleotide sequencerepresented by SEQ ID NO: 89, and adding sterilized distilled water to atotal amount of 50 μL. This reaction solution is maintained at 94° C.for 2 minutes, and further 35 cycles of incubation are repeated, eachcycle comprising maintaining a temperature at 94° C. for 15 seconds,then, at 68° C. for 5 minutes. The amplified DNA is purified from thePCR reaction solution using QIAquick PCR Purification Kit (QIAGEN)according to the instruction attached to the kit, and 3′A addition isperformed on the DNA according to the method described in Example 11(1). 3′A added DNA and the pCR2.1-TOPO cloning vector (Invitrogen) areligated according to the instruction attached to the cloning vector,after that, which is introduced into Escherichia coli JM109 (TaKaRa). Aplasmid DNA is purified from the resulting Escherichia coli transformantusing QIAprep Spin Miniprep Kit (QIAGEN). A nucleotide sequence isanalyzed using the resulting plasmid DNA as a template and using primersconsisting of the nucleotide sequence represented by any of SEQ ID NOs:28, 29, and the like according to the method described in Example 11(1).As a result, a nucleotide sequence of the 5′-terminal region including atranslation initiation codon of an os-1 homologous gene of Phytophthorainfestans, that is, gene of Phytophthora infestans encoding osmosensinghistidine kinase having no transmembrane region (PiOS1) can be read.

Further, a DNA having a nucleotide sequence extending to 3′ downstreamregion of the nucleotide sequence represented by SEQ ID NO: 89 iscloned. 1.0 μL of CDS-primer attached to the kit and 1.0 μL ofsterilized distilled water are mixed into 3 μL (200 ng) of the total RNAprepared in Example 10 (4), a temperature is maintained at 70° C. for 2minutes, and is maintained on ice for 2 minutes. 3′ RACE ready cDNA isprepared using the reaction solution as in preparation of 5′ RACE readycDNA. PCR amplifying 3′ downstream region is performed using this 3′RACE ready cDNA as a template. The PCR reaction solution is prepared bymixing 5.0 μL of 10× Advantage 2 buffer attached to the kit, 1.0 μL itof 10 mM dNTP Mix and 1.0 μL of 50× Advantage 2 polymerase Mix into 2.5μL of 5′ RACE ready cDNA, adding 5.0 μL of 10× Universal Primer A Mixattached to the kit, and 1.0 μL of a 10 μM solution of anoligonucleotide consisting of 20 to 30 bases selected from thenucleotide sequence represented by SEQ ID NO: 89 as primers, and addingsterilized distilled water to a total amount of 50 μL. This reactionsolution is subjected to repetition of 5 cycles of incubation, eachcycle comprising maintaining a temperature at 94° C. for 5 seconds,then, at 72° C. for 4 minutes, further repetition of 5 cycles ofincubation, each cycle comprising maintaining a temperature at 94° C.for 5 seconds, then, at 70° C. for 10 seconds then, at 72° C. for 4minutes, further repetition of 25 cycles of incubation, each cyclecomprising maintaining a temperature at 94° C. for 5 seconds, then, at68° C. for 10 seconds, then, at 72° C. for 4 minutes, followed bymaintaining a temperature at 72° C. for 7 minutes. The PCR reactionsolution and the pCR2.1-TOPO cloning vector (Invitrogen) are ligatedaccording to the instruction attached to the cloning vector, after that,which is introduced into Escherichia coli JM109 (TaKaRa). A plasmid DNAis purified from the resulting Escherichia coli transformant usingQIAprep Spin Miniprep Kit (QIAGEN). A nucleotide sequence is analyzedusing the resulting plasmid DNA as a template and using primersconsisting of the nucleotide sequence represented by any of SEQ ID NOs:28, 29, and the like according to the method described in Example 11(1). As a result, a nucleotide sequence of the 3′-terminal regionincluding a translation termination codon of a Phytophthora infestansPiOS1 gene is read.

By ligating all analyzed nucleotide sequences, full nucleotide sequenceof Phytophthora infestans PiOS-1 gene including nucleotide sequencerepresented by SEQ ID NO: 89 is confirmed.

(3) Isolation of Full Length Phytophthora infestans PiOS1 Gene

A DNA having a nucleotide sequence encoding an amino acid sequence ofPhytophthora infestans PiOS1 (hereinafter, referred to as PiOS-1 DNA) isamplified by PCR using the cDNA prepared in Example 10 (4) as atemplate. Using as primers an oligonucleotide comprising a nucleotidesequence in which a nucleotide sequence ACGACAGT is added to the5′-terminal end of a nucleotide sequence from the 5′-terminal end to the20^(th) base including the initiation codon of a nucleotide sequence ofPhytophthora infestans PiOS-1 gene obtained in Example 20 (2), and anoligonucleotide having a nucleotide sequence complementary to anucleotide sequence in which a nucleotide sequence AAGCTTCAG is added tothe 3′-terminal end of a nucleotide sequence of from the 3′-terminal endto the 20^(th) base including the termination codon of a nucleotidesequence of Phytophthora infestans PiOS-1 gene obtained in Example 20(2), a PCR is performed according to the method described in Example 11(3). DNA containing a nucleotide sequence encoding an amino acidsequence of Phytophthora infestans PiOS-1, and having a recognitionsequence of a restriction enzyme SpeI immediately before an initiationcodon, and having a recognition sequence of a restriction enzyme HindIIIimmediately after a termination codon is amplified. A part of the PCRreaction solution after the reaction is separated by 1% agarose gelelectrophoresis, and stained with ethidium bromide. It is confirmed thatthe about 4 kb PiOS-1 DNA is amplified.

Example 21 Construction of Expression Plasmid of Phytophthora infestansPiOS-1 Gene and Preparation of Transformed Budding Yeast

The PiOS-1 DNA is cloned into the pCR2.1-TOPO cloning vector(Invitrogen). An about 4 kb DNA (PiOS-1 DNA) is purified from the PCRreaction solution prepared in Example 20 (3) using QIAquick PCRPurification Kit (QIAGEN) according to the manual attached to the kit.3′A addition is performed on the about 4 kb purified DNA according tothe method described in Example 11 (3). The about 4 kb 3′A-added DNA(PiOS-1 DNA) and the pCR2.1-TOPO cloning vector (Invitrogen) are ligatedaccording to the instruction attached to the cloning vector, whereby,the plasmid pCRPiOS1 is constructed. A nucleotide sequence of theresulting plasmid is analyzed by the method described in Example 11 (1).As a primer, oligonucleotides consisting of the nucleotide sequencerepresented by any of SEQ ID NOs: 28 and 29 are used. As a result, it isconfirmed that the plasmid pCRPiOS1 is a plasmid harboring PiOS-1 DNAcontaining the nucleotide sequence represented by SEQ ID NO: 89.

The Phytophthora infestans PiOS-1 gene contained in the thus preparedplasmid pCR PiOS1 is cloned into a shuttle vector p415ADH replicable inyeast and Escherichia coli, whereby, an expression plasmid isconstructed. The plasmid pCRPiOS1 is digested with restriction enzymesSpeI and HindIII and, on the other hand, the shuttle vector p415ADH isalso digested with restriction enzymes SpeI and HindIII. These areseparated by 0.8% agarose gel electrophoresis, respectively, thereafter,a part of the gel containing the PiOS-1 DNA digested with restrictionenzymes SpeI and HindIII and the shuttle vector p415ADH digested withSpeI and HindIII is excised, and the PiOS-1 DNA and the shuttle vectorare recovered from the gel using QIAquick Gel Extraction Kit (QUAGEN)according to the manual attached to the kit. Using Ligation Kit Ver. 2(TaKaRa) according to the manual attached to the kit, the PiOS-1 DNA isinserted between SpeI site and HindIII site in the multicloning site ofthe shuttle vector, whereby, the expression plasmid pADHPiOS1 isconstructed. A nucleotide sequence of the resulting expression plasmidis analyzed according to the method described in Example 11 (1). As aprimer, oligonucleotides consisting of the nucleotide sequencerepresented by any of SEQ ID NOs: 28 and 29 are used. As a result, it isconfirmed that the expression plasmid pADHPiOS1 is a plasmid harboringthe PiOS-1 DNA containing the nucleotide sequence represented by SIQ IDNO: 89.

The prepared expression plasmid pADH PiOS1 is gene-introduced intobudding yeast AH22 strain and TM182 strain according to the methoddescribed in Example 2. By utilizing disappearance of leucine auxotrophyin the resulting transformed budding yeast, the transformed buddingyeast AH22 strain (AH22-PiOS1) is selected on a Glu-Leu agar medium, andthe transformed budding yeast TM182 strain (TN182-PiOS1) is selected ona Gal-Ura-Leu agar medium. It is confirmed that the resultingTM182-PiOS1 grows even when transplanted to a Glu-Ura-Leu medium.

Example 22 Antifungal Compound Sensitivity Test of Transformed BuddingYeast TM182-PiOS1

The transformed budding yeast AH22-PiOS1 prepared in Example 21 iscultured while shaking at 30° C. in a Glu-Leu medium. As a control, theAH22 strain is similarly cultured while shaking at 30° C. in a Glumedium. The absorbance at 600 nm of each of grown transformed buddingyeasts in a cell suspension is measured, and a cell suspension dilutedwith sterilized distilled water to the absorbance of 0.1 is prepared.Further, a cell suspension in which the aforementioned suspension of thetransformed budding yeast AH22-PiOS1 is diluted 50-fold with a Glu-Leumedium, and a cell suspension in which the aforementioned suspension ofthe AH22 strain is diluted 50-fold with a Glu medium are prepared.

A solution in which each of Compounds (1) to (7) is dissolved indimethylsulfoxide (DMSO) is prepared, and two microplates are preparedin which each 1.0 μL per well of each of the Compound Solution and DMSOas a control are dispensed into two wells. In one microplate among them,each 100 μL of cell suspensions of the transformed budding yeastAH22-PiOS1 which has been prepared by dilution as described above isdispensed, and is cultured by allowing to stand at 30° C. In anothermicroplate, each 100 μL of cell suspensions of the control yeast AH22strain which has been prepared by dilution as described above isdispensed, and is cultured by allowing to stand at 30° C. Afterculturing, the absorbance at 600 nm of each well is measured with amicroplate reader.

Similarly, the transformed budding yeast TM182-PiOS1 prepared in Example21 is cultured at 30° C. in a Glu-Ura-Leu medium. The absorbance at 600nm of a cell suspension of the grown transformed budding yeast ismeasured, and a cell suspension diluted with sterilized distilled waterto the absorbance of 0.1 is prepared. Further, a cell suspension inwhich the aforementioned cell suspension of the transformed buddingyeast TM182-PiOS1 is diluted 50-fold with a Glu-Ura-Leu medium and, as acontrol, a cell suspension in which the aforementioned cell suspensionis diluted 50-fold with a Gal-Ura-Leu medium are prepared.

A solution in which each of Compounds (1) to (7) is dissolved indimethylsulfoxide (DMSO) is dissolved is prepared, and two microplatesare prepared in which each 1.0 μL per well of the Compound solution andDMSO as a control are dispensed. In one microplate among them, each 100μL of cell suspensions of the transformed budding yeast TM182-PiOS1which has been prepared by dilution with a Glu-Ura-Leu medium asdescribed above is dispensed, and is cultured by allowing to stand at30° C. In another microplate as described above, as a control, each 100μL of cell suspensions of the transformed budding yeast TM182-PiOS1which has been prepared by dilution with a Gal-Ura-Leu medium isdispensed, and is cultured by allowing to stand at 30° C. Afterculturing, the absorbance at 600 nm of each well is measured with amicroplate reader.

It is confirmed that an inhibiting degree of growth of the transformedbudding yeast TM182-PiOS1 by each test substance is greater than aninhibiting degree of growth of the transformed budding yeast AH22-PiOS1by each test substance, and the transformed budding yeast TM182-PiOS1 isa transformed cell with the enhanced sensitivity to an antifungalcompound as compared with the transformed budding yeast AH2-PiOS1.

The compositions of media used in the present invention are describedbelow.

(a) Glu-Medium

Becto-yeast nitrogen base without amino acids 6.7 g, Glucose 20 g,Drop-out mix (1) 2.0 g, Distilled water 1000 ml

(b) Glu-Leu Medium

Bacto-yeast nitrogen base without amino acids 6.7 g, Glucose 20 g,Drop-out mix (2) 2.0 g, Distilled water 1000 ml

(c) Glu-Ura-Leu Medium

Bacto-yeast nitrogen base without amino acids 6.7 g, Glucose 20 g,Drop-out mix (3) 2.0 g,

Distilled water 1000 ml

(d) Gal-Ura-Leu Medium

Bacto-yeast nitrogen base without amino acids 6.7 g,

Galactose 20 g Drop-out mix (3) 2.0 g,

Distilled water 1000 ml

Drop-out mix (1):

Adenine 0.5 g, Lysine 2.0 g, Alanine 2.0 g, Methionine 2.0 g, Arginine2.0 g, para-Aminobenzoic acid 0.2 g, Asparagine 2.0 g. Phenylalanine 2.0g, Aspartic acid 2.0 g, Proline 2.0 g, Cysteine 2.0 g, Serine 2.0 g,Glutamine 2.0 g, Threonine 2.0 g, Glutamic acid 2.0 g, Tryptophan 2.0 g,Glycine 2.0 g, Tyrosine 2.0 g, Histidine 2.0 g, Valine 2.0 g, Inositol2.0 g, Isoleucine 2.0 g, Uracil 2.0 g, Leucine 10.0 g, Distilled water1000 ml

Drop-out mix (2): Drop-out mix (1) except for leucine (10.0 g)

Drop-out mix (3): Drop-out mix (1) except for uracil (2.0 g) and leucine(10.0 g)

(e) Glu-agar Medium

Solid medium in which 2% (W/V) agar is added to a medium (a)

(f) Glu-Leu agar Medium

Solid medium in which 2% (W/V) agar is added to a medium (b)

(g) Glu-Ura-Leu Agar Medium

Solid medium in which 2% (W/V) agar is added to a medium (c)

(h) Gal-Ura-Leu Agar Medium

Solid medium in which 2% (W/V) agar is added to a medium (d)

Free text in Sequence Listing

SEQ ID NO:3

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Designed oligonucleotide primer for PCR

1. A transformed cell in which a polynucleotide encoding an osmosensing histidine kinase having no transmembrane region is introduced in a functional form into a cell that is deficient in at least one hybrid-sensor kinase, wherein the osmosensing histidine kinase having no transmembrane region has an amino acid sequence homology of 98% or more to the amino acid sequence of SEQ ID NO:
 55. 2. The transformed cell according to claim 1, wherein the polynucleotide complements the hybrid-sensor kinase deficiency.
 3. The transformed cell according to claim 1, wherein the cell is a microorganism cell.
 4. The transformed cell according to claim 1, wherein the cell is a budding yeast cell.
 5. The transformed cell according to claim 1, wherein the osmosensing histidine kinase having no transmembrane region has the amino acid sequence of SEQ ID NO:
 55. 6. The transformed cell according to claim 1, wherein the polynucleotide has the nucleotide sequence of SEQ ID NO:
 56. 7. A method of assaying the antifungal activity of a substance, which comprises: (1) culturing the transformed cell as defined in claim 1 in the presence of a test substance; (2) measuring an amount of intracellular signal transduction from the osmosensing histidine kinase having no transmembrane region or an index value having the correlation therewith; and (3) assessing the antifungal activity of the test substance based on a difference between an amount of intracellular signal transduction or an index value having the correlation therewith measured in (2) and a control.
 8. The method of assaying according to claim 7, wherein the amount of intracellular signal transduction from the osmosensing histidine kinase having no transmembrane region or the index value having the correlation therewith is an amount of growth of the transformed cell.
 9. A method of searching for a potent antifungal compound, which comprises selecting an antifungal compound based on the antifungal activity assessed in the assaying method as defined in claim
 7. 10. An isolated polynucleotide encoding an amino acid sequence selected from the group consisting of: (a) an amino acid sequence of an osmosensing histidine kinase having no transmembrane region, which has amino acid sequence homology of 98% or more to the amino acid sequence of any of SEQ ID NO: 55 and (b) the amino acid sequence of SEQ ID NO:
 55. 11. The polynucleotide according to claim 10, which encodes the amino acid sequence of SEQ ID NO:
 55. 12. The polynucleotide according to claim 10, which has the nucleotide sequence of SEQ ID NO:
 56. 13. The transformed cell according to claim 1, wherein the polynucleotide encodes an osmosensing histidine kinase having no transmembrane region is obtained from Mycospharella tritici. 